Difference between revisions of "Berk2010-Christoph"

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(Christoph Neyer 14:22, 26 July 2010 (EDT))
(Christoph Neyer 14:52, 28 July 2010 (EDT))
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Here is the RE map for parts from Pippin: <br>
 
Here is the RE map for parts from Pippin: <br>
 
Gel: <br>
 
Gel: <br>
[[Image:Stage_4_with_Stage_3_cathups_from_Pippin.jpg|500px]]
+
[[Image:Stage_4_with_Stage_3_cathups_from_Pippin.jpg|500px]] <br>
 
Layout: <br>
 
Layout: <br>
 
{| {{table}}
 
{| {{table}}
Line 64: Line 64:
 
| iGEM10_110 in KA||39||40||iGEM10_110||3218||4490||1605
 
| iGEM10_110 in KA||39||40||iGEM10_110||3218||4490||1605
 
|}
 
|}
 +
 
==[[User:Christoph Neyer|Christoph Neyer]] 14:22, 26 July 2010 (EDT)==
 
==[[User:Christoph Neyer|Christoph Neyer]] 14:22, 26 July 2010 (EDT)==
 
Assembled Stage 3 parts and some stage 2 catchups.<br>
 
Assembled Stage 3 parts and some stage 2 catchups.<br>

Revision as of 10:55, 28 July 2010

Contents

TO DO

  • Organize catchups for stage 2.
  • Assemble Stage 2 catchups.
  • Check cotranformations for stage 2.

Christoph Neyer 14:52, 28 July 2010 (EDT)

Assembled Stage 4 Parts and some Stage 3 catchups.
Here is the RE map for parts from Pippin:
Gel:
Stage 4 with Stage 3 cathups from Pippin.jpg
Layout:

' ' ' ' ' ' '
Clones A and B are Gel #1 Clones C and D are on Gel #2
Stage 4 parts + Stage 3 Catchups from Pippin Lanes (Clones A and C) Lanes (Clones B and D) Part Name Part size Eco/xho Frags eco/Xho Frags
iGEM10_117 in CA 1 2 iGEM10_117 6246 7518 1473
iGEM10_118 in CA 3 4 iGEM10_118 4138 5410 1473
iGEM10_119 in CA 5 6 iGEM10_119 6645 7917 1473
iGEM10_120 in CA 7 8 iGEM10_120 4138 5410 1473
iGEM10_121 in CA 9 10 iGEM10_121 6645 7917 1473
iGEM10_132 in CA 11 12 iGEM10_132 3739 5011 1473
iGEM10_133 in CA 13 14 iGEM10_133 3697 4969 1473
iGEM10_136 in CA 15 16 iGEM10_136 4096 5368 1473
iGEM10_088 in CA 17 18 iGEM10_088 1096 2368 1473
iGEM10_088 in CA 19 20 iGEM10_088 1096 2368 1473
iGEM10_127 in CK 21 22 iGEM10_127 4187 5387 1473
iGEM10_128 in CK 23 24 iGEM10_128 5676 6876 1473
iGEM10_129 in CK 25 26 iGEM10_129 5740 6940 1473
iGEM10_138 in CA 27 28 iGEM10_138 4096 5368 1473
iGEM10_139 in CA 29 30 iGEM10_139 3474 4746 1473
iGEM10_103 in KA 31 32 iGEM10_103 5725 6997 1605
iGEM10_103 in KA 33 34 iGEM10_103 5725 6997 1605
iGEM10_104 in KA 35 36 iGEM10_104 4869 6141 1605
iGEM10_110 in KA 37 38 iGEM10_110 3218 4490 1605
iGEM10_110 in KA 39 40 iGEM10_110 3218 4490 1605

Christoph Neyer 14:22, 26 July 2010 (EDT)

Assembled Stage 3 parts and some stage 2 catchups.
Here is the RE map for the parts from Gandalf:
Gel:
Stage 3 with stage 2 catchups RE map of Gandalf.jpg
Layout:

' Clones A and B are Gel #1 Clones C and D are on Gel #2 ' ' ' '
Stage 3 parts + Stage 2 Cathups from Gandalf Lanes (Clones A and C) Lanes (Clones B and D) Part Name Part size Eco/xho Frags eco/Xho Frags
iGEM10_105 in CA 1 2 iGEM10_105 2331 3603 1473
iGEM10_107 in CA 3 4 iGEM10_107 3169 4441 1473
iGEM10_109 in CA 5 6 iGEM10_109 3233 4505 1473
iGEM10_088 in AC 7 8 iGEM10_088 1096 2162 1679
iGEM10_084 in KC 9 10 iGEM10_084 2154 3220 1605
iGEM10_101 in KA 11 12 iGEM10_101 4911 6183 1605
iGEM10_102 in KA 13 14 iGEM10_102 2404 3676 1605
iGEM10_108 in AK 15 16 iGEM10_108 1018 2218 1680
iGEM10_111 in KA 17 18 iGEM10_111 2362 3634 1605
iGEM10_114 in KA 19 20 iGEM10_114 1740 3012 1605
iGEM10_112 in CA 21 22 iGEM10_112 2030 3302 1473
iGEM10_113 in CA 23 24 iGEM10_113 2049 3321 1473
iGEM10_115 in CA 25 26 iGEM10_115 1966 3238 1473
iGEM10_116 in CA 27 28 iGEM10_116 1985 3257 1473
iGEM10_085 in CA 29 30 iGEM10_085 3571 4843 1473
iGEM10_087 in CA 31 32 iGEM10_087 3564 4836 1473
iGEM10_097 in CA 33 34 iGEM10_097 2278 3550 1473

Christoph Neyer 15:47, 22 July 2010 (EDT)

Did:

  • Miniprep Stage 1 parts
  • Miniprep Stage 2 parts
  • Pick colonies for iGEM10_064 in KC and iGEM10_65 in CK, and sbb18 in KA.

Christoph Neyer 16:42, 22 July 2010 (EDT)

Stage 1 Retransformations from Marley RE map:
Gel Part A:
Stage 1 retransformations gel part A.jpg
Gel Part B:
Stage 1 retransformations gel part B.jpg
Small Gel:
Stage 1 retransformations small gel.jpg

Layout:

Stage 1 Re-transforms From Marley Lanes (Clones A) Lanes (Clones B) Part Name Part size Eco/xho Frags eco/Xho Frags
iGEM10_077 in CA 1 2 iGEM10_077 1879 3151 1473
iGEM10_078 in CA 3 4 iGEM10_078 1522 2794 1473
iGEM10_079 in CA 5 6 iGEM10_079 1117 2389 1473
iGEM10_056 in AC 7 8 iGEM10_056 82 1148 1679
iGEM10_061 in CA 9 10 iGEM10_061 1064 2336 1473
iGEM10_063 in CA 11 12 iGEM10_063 1057 2329 1473
iGEM10_066 in CA 13 14 iGEM10_066 80 1352 1473
iGEM10_062 in AC 15 16 iGEM10_062 279 1345 1679
iGEM10_054 in CA 17 18 iGEM10_054 1335 2607 1473
iGEM10_067 in CA 19 20 iGEM10_067 157 1429 1473
iGEM10_068 in AK 21 22 iGEM10_068 771 1971 1680
iGEM10_055 in KA 23 24 iGEM10_055 1464 2736 1605
iGEM10_055 in KA 25 26 iGEM10_055 1464 2736 1605
iGEM10_065 in CK 27 28 iGEM10_065 2554 3754 1473
iGEM10_057 in CK 29 30 iGEM10_057 858 2058 1473
Ladder Ladder
iGEM10_071 in KC 31 32 iGEM10_071 1909 2975 1605
iGEM10_072 in KC 33 34 iGEM10_072 697 1763 1605
iGEM10_061 in CK 35 36 iGEM10_061 1064 2264 1473
iGEM10_063 in CK 37 38 iGEM10_063 1057 2257 1473
iGEM10_073 in CK 39 40 iGEM10_073 996 2196 1473
iGEM10_074 in CK 41 42 iGEM10_074 490 1690 1473
EMPTY 43 44 EMPTY #N/A #N/A #N/A
iGEM10_054 in CK 45 46 iGEM10_054 1335 2535 1473
Small gel
iGEM10_076 in AK 1 2 iGEM10_076 1878 3078 1680
iGEM10_057 in CA 3 4 iGEM10_057 858 2130 1473
iGEM10_060 in AC 5 6 iGEM10_060 366 1432 1679
iGEM10_075 in CK 7 8 iGEM10_075 490 1690 1473
iGEM10_065 in CK 9 10 iGEM10_065 2554 3754 1473

Christoph Neyer 15:46, 22 July 2010 (EDT)

Stage 2 Parts RE map from Frodo:
Gel:
Stage 2 parts Re map from Frodo 7-22-10.jpg
Layout:

Stage 2 parts from Frodo Clones A and B are Gel #1 Clones C and D are on Gel #2 ' ' ' '
Lanes (Clones A and C) Lanes (Clones B and D) Part Name Part size Eco/xho Frags eco/Xho Frags
iGEM10_080 in KC 1 2 iGEM10_080 1546 2612 1605
iGEM10_082 in CK 3 4 iGEM10_082 1734 2934 1473
iGEM10_083 in CK 5 6 iGEM10_083 1734 2934 1473
iGEM10_084 in KC 7 8 iGEM10_084 2154 3220 1605
iGEM10_086 in KC 9 10 iGEM10_086 1305 2371 1605
iGEM10_081 in CA 11 12 iGEM10_081 3365 4637 1473
iGEM10_093 in CA 13 14 iGEM10_093 1043 2315 1473
iGEM10_094 in CA 15 16 iGEM10_094 2278 3550 1473
iGEM10_095 in CA 17 18 iGEM10_095 1954 3226 1473
Ladder Ladder #N/A #N/A #N/A
iGEM10_096 in CA 19 20 iGEM10_096 1516 2788 1473
iGEM10_098 in CA 21 22 iGEM10_098 1954 3226 1473
iGEM10_099 in CA 23 24 iGEM10_099 1516 2788 1473
iGEM10_100 in CA 25 26 iGEM10_100 1087 2359 1473
iGEM10_089 in KA 27 28 iGEM10_089 1925 3197 1605
iGEM10_091 in KA 29 30 iGEM10_091 1898 3170 1605
iGEM10_092 in KA 31 32 iGEM10_092 1989 3261 1605
iGEM10_090 in CK 33 34 iGEM10_090 928 2128 1473

Christoph Neyer 19:08, 21 July 2010 (EDT)

Stage 1 parts grew up last night. Most things grew. Picked 4 colonies for most of the parts from stage 2 to miniprep tomorrow.
Yesterday we plated all the stage 1 parts and grew them up on plates. Today we picked 2 colonies for each to miniprep tomorrow.
Reassembled igem10_064 in KC and retransformed igem10_065 CK (Also picked colonies from an old plate).
Also retransformed Sbb18 in KA Lefty because miniprep is weak.

Christoph Neyer 15:38, 20 July 2010 (EDT)

RE map of Ophelia columns 1-3:
RE map of Ophelia.jpg
Layout:

' Lane # Part Name Part size Eco/xho Frags eco/Xho Frags
iGEM10_076 in AK 1 iGEM10_076 1878 3078 1680
iGEM10_077 in CA 2 iGEM10_077 1879 3151 1473
iGEM10_078 in CA 3 iGEM10_078 1522 2794 1473
iGEM10_079 in CA 4 iGEM10_079 1117 2389 1473
iGEM10_055 in KA 5 iGEM10_055 1464 2736 1605
iGEM10_068 in AK 6 iGEM10_068 771 1971 1680
iGEM10_065 in CK 7 iGEM10_065 2554 3754 1473
iGEM10_056 in AC 8 iGEM10_056 82 1148 1679
iGEM10_057 in CA 9 iGEM10_057 858 2130 1473
iGEM10_060 in AC 10 iGEM10_060 366 1432 1679
iGEM10_061 in CA 11 iGEM10_061 1064 2336 1473
iGEM10_063 in CA 12 iGEM10_063 1057 2329 1473
iGEM10_057 in CK 13 iGEM10_057 858 2058 1473
iGEM10_061 in CK 14 iGEM10_061 1064 2264 1473
iGEM10_063 in CK 15 iGEM10_063 1057 2257 1473
iGEM10_071 in KC 16 iGEM10_071 1909 2975 1605
iGEM10_072 in KC 17 iGEM10_072 697 1763 1605
iGEM10_073 in CK 18 iGEM10_073 996 2196 1473
iGEM10_074 in CK 19 iGEM10_074 490 1690 1473
iGEM10_075 in CK 20 iGEM10_075 490 1690 1473
iGEM10_055 in KA 21 iGEM10_055 1464 2736 1605
iGEM10_054 in CA 22 iGEM10_054 1335 2607 1473
iGEM10_067 in CA 23 iGEM10_067 157 1429 1473
iGEM10_066 in CA 24 iGEM10_066 80 1352 1473

Christoph Neyer 15:47, 19 July 2010 (EDT)

RE mapping of Stage 1 Catchups from Guildenstern Columns 5+7:
Gel:
Stage 1 Round 4 Parts RE map Catchups from Guildenstern columns 5+7.jpg Layout:

' ' ' Part Name Part size Eco/xho Frags eco/Xho Frags
iGEM10_062 in AC 1,2 3,4 iGEM10_062 279 1345 1679
iGEM10_079 in CA 5 6 iGEM10_079 1117 2389 1473
iGEM10_065 in CK 7 8 iGEM10_065 2554 3754 1473

Christoph Neyer 19:57, 18 July 2010 (EDT)

RE mapping Stage 1 Parts from Rosencrantz and Guildenstern:

  • Gel # 1 (Clones A+B from Rosencrantz):
    • Part A:
    • Stage 1 Round 4 Parts RE map Gel -1 A, parts from Rosencrantz.jpg
    • Part B:
    • Stage 1 Round 4 Parts RE map Gel -1 B, parts from Rosencrantz.jpg
  • Gel # 2 (Clones A+B from Rosencrantz):
    • Part A:
    • Stage 1 Round 4 Parts RE map Gel -2 A, parts from Rosencrantz.jpg
    • Part B:
    • Stage 1 Round 4 Parts RE map Gel -2 B, parts from Rosencrantz.jpg
  • Gel # 3 (Guildenstern Columns 1+3):
    • Stage 1 Round 4 Parts RE map Gel -3, parts from Guildenstern.jpg

Layout:

Clones A and B are Gel #1 ' ' ' ' ' '
Clones C and D are on Gel #2
Lanes Clone A,C Lanes Clone B,D Part Name Part size Eco/xho Frags eco/Xho Frags
iGEM10_076 in AK 1 2 iGEM10_076 1878 3078 1680
iGEM10_077 in CA 3 4 iGEM10_077 1879 3151 1473
iGEM10_078 in CA 5 6 iGEM10_078 1522 2794 1473
iGEM10_079 in CA 7 8 iGEM10_079 1117 2389 1473
iGEM10_055 in KA 9 10 iGEM10_055 1464 2736 1605
iGEM10_068 in AK 11 12 iGEM10_068 771 1971 1680
iGEM10_064 in KC 13 14 iGEM10_064 996 2062 1605
iGEM10_065 in CK 15 16 iGEM10_065 2554 3754 1473
iGEM10_056 in AC 17 18 iGEM10_056 82 1148 1679
iGEM10_057 in CA 19 20 iGEM10_057 858 2130 1473
iGEM10_060 in AC 21 22 iGEM10_060 366 1432 1679
iGEM10_062 in AC 23 24 iGEM10_062 279 1345 1679
iGEM10_066 in CA 25 26 iGEM10_066 80 1352 1473
iGEM10_061 in CA 27 28 iGEM10_061 1064 2336 1473
iGEM10_063 in CA 29 30 iGEM10_063 1057 2329 1473
iGEM10_054 in CK 31 32 iGEM10_054 1335 2535 1473
iGEM10_057 in CK 33 34 iGEM10_057 858 2058 1473
iGEM10_061 in CK 35 36 iGEM10_061 1064 2264 1473
iGEM10_063 in CK 37 38 iGEM10_063 1057 2257 1473
iGEM10_071 in KC 39 40 iGEM10_071 1909 2975 1605
iGEM10_072 in KC 41 42 iGEM10_072 697 1763 1605
iGEM10_073 in CK 43 44 iGEM10_073 996 2196 1473
iGEM10_074 in CK 45 46 iGEM10_074 490 1690 1473
iGEM10_075 in CK 47 48 iGEM10_075 490 1690 1473
Small Gel
iGEM10_055 in KA 1 2 iGEM10_055 1464 2736 1605
iGEM10_054 in CA 1,2 3,4 iGEM10_054 1335 2607 1473
iGEM10_067 in CA 5,6 7,8 iGEM10_067 157 1429 1473
iGEM10_066 in CA 9,10 11,12 iGEM10_066 80 1352 1473

Christoph Neyer 22:00, 14 July 2010 (EDT)

RE map of KA transfers:
Re map of KA transfers for stage 1.jpg
Layout:

' Lane Lane part Part size Eco/xho Frags eco/Xho Frags
sbb19 in KA 1 2 sbb19 597 1869 1605
sbb18 in KA 3 4 sbb18 399 1671 1605
sbb10 in KA 5 6 sbb10 1026 2298 1605
sbb42 in KA 7 8 sbb42 91 1363 1605
sbb04 in KA 9 10 sbb04 1788 3060 1605
sbb06 in KA 11 12 sbb06 241 1513 1605
sbb04 in KA 13 14 sbb04 1788 3060 1605
Bjh2245 in KA 15 16 Bjh2245 97 1369 1605
Bjh1906 in KA 17 18 Bjh1906 47 1319 1605
sbb19 in KA 19 20 sbb19 597 1869 1605
sbb12 in KA 21 22 sbb12 234 1506 1605
sbb09 in KA 23 24 sbb09 35 1307 1605
sbb10 in KA 25 26 sbb10 1026 2298 1605
sbb42 in KA 27 28 sbb42 91 1363 1605
Bjh2294 in KA 29 30 Bjh2294 2507 3779 1605
sbb07 in KA 31 32 sbb07 1431 2703 1605

Christoph Neyer 16:08, 14 July 2010 (EDT)

Checking some of the parts from Oliver twist:
Re map Double Checking oliver twist.jpg
Layout:

Lane # ' Plate Location part Part size Eco/xho Frags eco/Xho Frags
1 sbb15 in AC Future A1 sbb15 90 1156 1679
3 sbb43 in AK Future A4 sbb43 100 1300 1680
5 Bjh1906 in AK Future B4 Bjh1906 47 1247 1680
7 sbb13 in AC Future B7 sbb13 1818 2884 1679
9 Bjh2294 in AK Future F10 Bjh2294 2507 3707 1680
11 Bjh1858 in CA Future A12 Bjh1858 33 1305 1473
2 sbb15 in AC Tiny Tim A1 sbb15 90 1156 1679
4 sbb43 in AK Tube C na sbb43 100 1300 1680
6 Bjh1906 in AK Cat A1 Bjh1906 47 1247 1680
8 sbb13 in AC Tiny Tim C7 sbb13 1818 2884 1679
10 Bjh2294 in AK Cat A5 Bjh2294 2507 3707 1680
12 Bjh1858 in CA Scrooge A12 Bjh1858 33 1305 1473

Christoph Neyer 18:05, 13 July 2010 (EDT)

Rechecked pMLL7 and pMLL4 for methylated parts:
Stage 0 vector rechecks.jpg
Layout:

Lane # Part in vector opposite vector Part Vector Part Location in Tiny Tim Enzymes Expected bands Expected bands Wrong Bands Wrong Bands
1 Bjh2294 in AK pMLL6-KA Bjh2294 pMLL7-AK Bjh2294 Get from Chesire cat A 5 EcoRI/Bgl1 4554 830 3479 1905
2 Bjh1906 in CA pMLL4-AC Bjh1906 pMLL9-CA Bjh1906 A6 EcoRI/Bgl1 1773 1019 1962 830
3 sbb06 in AK pMLL6-KA sbb06 pMLL7-AK sbb06 G10 EcoRI/Bgl1 2288 830 1905 1213
4 sbb44 in CA pMLL4-AC sbb44 pMLL9-CA sbb44 C6 EcoRI/Bgl1 1795 1773 2738 830

Christoph Neyer 23:23, 12 July 2010 (EDT)

Stage 1 Round 3 RE map of minipreps for parts not involving KA inputs. Located on Oliver Twist. Gel Part A:
Stage 1 Round 3 Minipreps RE map from Oliver Twist Part A.jpg
Gel Part B:
Stage 1 Round 3 Minipreps RE map from Oliver Twist Part B.jpg
Layout:

RE Map of Oliver Twist Part A Lane # Part B Lane # Part C Lane # Part D Lane # Part Name Part size Lefty size Righty Size Eco/xho Frags eco/Xho Frags Analysis A Analysis B Re-run/re-pick?
iGEM10_076 in AK 1 2 23 24 iGEM10_076 1878 1845 33 3078 1680
iGEM10_055 in KA 3 4 25 26 iGEM10_055 1464 1431 33 2736 1605
iGEM10_068 in AK 5 6 27 28 iGEM10_068 771 681 90 1971 1680
iGEM10_065 in CK 7 8 29 30 iGEM10_065 2554 47 2507 3754 1473
iGEM10_073 in CK 9 10 31 32 iGEM10_073 996 399 597 2196 1473
iGEM10_054 in CK 11 12 33 34 iGEM10_054 1335 1244 91 2535 1473
iGEM10_057 in CK 13 14 35 36 iGEM10_057 858 823 35 2058 1473
iGEM10_061 in CK 15 16 37 38 iGEM10_061 1064 823 241 2264 1473
iGEM10_063 in CK 17 18 39 40 iGEM10_063 1057 823 234 2257 1473
iGEM10_074 in CK 19 20 41 42 iGEM10_074 490 91 399 1690 1473
iGEM10_055 in KA 21 22 43 44 iGEM10_055 1464 1431 33 2736 1605

Christoph Neyer 19:24, 12 July 2010 (EDT)

Col PCR Stage 1 Round 3:
Part A:
Col PCR Gel -1 Part A.jpg
Part B:
Col PCR Gel -1 Part B.jpg
Part C:
Col PCR Gel -2.jpg

Christoph Neyer 16:41, 12 July 2010 (EDT)

Checking Vector Backbones Gel:
Checking Vector Backbones.jpg

Lane # Part in vector Vector + Ebid Location in Romeo Enzymes Expected bands Expected bands Wrong Bands Wrong Bands
1 sbb14 in AC 4 + 17 E8 EcoRI/Bgl1 3760 830 2817 1773
2 ig114 in CK 5 + 8 D5 EcoRI/ScaI 3221 694 2146 1769
3 sbb06 in KA 6 + 5 F1 EcoRI/Bgl1 1905 1213 2288 830
4 sbb17 in AK 7 + 14 E4 EcoRI/Bgl1 2446 830 1905 1371
5 sbb07 in KC 8 + 4 F6 EcoRI/ScaI 2333 1769 3408 694
6 sbb18 in CA 9 + 13 G9 EcoRI/Bgl1 1773 1371 2314 830

Christoph Neyer 21:25, 7 July 2010 (EDT)

Did colony PCR of Stage 1 Round 2assembly parts.

  • Gel #1
    • Part A:
      Stage 1 Col PCR Gel 1 Part A.jpg
    • Part B:
      Stage 1 Col PCR Gel 1 Part B.jpg
  • Gel #2
    • Part A:
      Stage 1 Col PCR Gel 2 Part A.jpg
    • Part B:
      Stage 1 Col PCR Gel 2 Part B.jpg



Layout for both gels:

' Lane ' '
Clone 1,3 Clone 2,4 Expected Band
iGEM10_076 in AK 1 2 2078
iGEM10_055 in KA 3 4 1664
iGEM10_068 in AK 5 6 971
iGEM10_064 in KC 7 8 1196
iGEM10_065 in CK 9 10 2754
iGEM10_056 in AC 11 12 282
iGEM10_057 in CA 13 14 1058
iGEM10_060 in AC 15 16 566
iGEM10_062 in AC 17 18 479
iGEM10_066 in CA 19 20 280
iGEM10_061 in CA 21 22 1264
iGEM10_063 in CA 23 24 1257
iGEM10_054 in CK 25 26 1535
iGEM10_057 in CK 27 28 1058
iGEM10_061 in CK 29 30 1264
iGEM10_063 in CK 31 32 1257
iGEM10_071 in KC 33 34 2109
iGEM10_072 in KC 35 36 897
iGEM10_073 in CK 37 38 1196
iGEM10_074 in CK 39 40 690
iGEM10_075 in CK 41 42 690
iGEM10_055 in KA 43 44 1664
iGEM10_054 in CA 45 46 1535
iGEM10_067 in CA 47 48 357

Christoph Neyer 19:35, 7 July 2010 (EDT)

Col PCR of oparts 77-79:
Stage 1 Col PCR Parts 77-79 7-7-10.jpg

Christoph Neyer 21:47, 6 July 2010 (EDT)

Did round 1 of 2ab assembly today. Here's the gel for ColPCR of 7 with 10 into Lefty strain.
7 with 10 redo col PCR.jpg

Christoph Neyer 16:31, 5 July 2010 (EDT)

Restriction map of Methylated Parts for stage 0 reruns:
Stage 0 mehtylated RE map reruns.jpg
Layout:

Re-runs ' ' ' ' ' '
sbb43 in AK 1 2 sbb43 100 1300 1680
ig114 in CA 3 4 ig114 1244 2516 1473
Bjh1858 in CK 5 6 Bjh1858 33 1233 1473
sbb07 in KA 7 8 sbb07 1431 2703 1605
sbb20 in AK 9 10 sbb20 597 1797 1680

Christoph Neyer 20:41, 2 July 2010 (EDT)

Restriction maps of Methylated Parts for Stage 0:

  • Lefty methylations:
    • Stage 0 mehtylated RE map part 1A.jpg
    • Stage 0 mehtylated RE map part 1B.jpg
    • Layout:
' Part A Lane # Part B Lane # Part Name Part size Eco/xho Frags eco/Xho Frags
sbb15 in AC 1 2 sbb15 90 1156 1679
sbb14 in AC 3 4 sbb14 1845 2911 1679
Bjh1882 in AC 5 6 Bjh1882 681 1747 1679
sbb19 in KA 7 8 sbb19 597 1869 1605
sbb18 in KA 9 10 sbb18 399 1671 1605
sbb10 in KA 11 12 sbb10 1026 2298 1605
sbb42 in KA 13 14 sbb42 91 1363 1605
sbb04 in KA 15 16 sbb04 1788 3060 1605
sbb42 in CK 17 18 sbb42 91 1291 1473
sbb37 in CK 19 20 sbb37 41 1241 1473
Bjh1858 in CK 21 22 Bjh1858 33 1233 1473
ig114 in CK 23 24 ig114 1244 2444 1473
Bca1091 in CK 25 26 Bca1091 60 1260 1473
sbb44 in CK 27 28 sbb44 823 2023 1473
sbb43 in AK 29 30 sbb43 100 1300 1680
Bjh1906 in AK 31 32 Bjh1906 47 1247 1680
sbb14 in KC 33 34 sbb14 1845 2911 1605
sbb13 in KC 35 36 sbb13 1818 2884 1605
sbb07 in KC 37 38 sbb07 1431 2497 1605
Bjh1906 in CA 39 40 Bjh1906 47 1319 1473
sbb18 in CA 41 42 sbb18 399 1671 1473
sbb44 in CA 43 44 sbb44 823 2095 1473
sbb42 in CA 45 46 sbb42 91 1363 1473
ig114 in CA 47 48 ig114 1244 2516 1473
  • Righty Methylations:
    • Stage 0 mehtylated RE map part 2A.jpg
    • Stage 0 mehtylated RE map part 2B.jpg
    • Layout:
' Part A Lane # Part B Lane # Part Name Part size Eco/xho Frags eco/Xho Frags
sbb43 in AC 1 2 sbb43 100 1166 1679
sbb13 in AC 3 4 sbb13 1818 2884 1679
sbb20 in AC 5 6 sbb20 597 1663 1679
sbb06 in KA 7 8 sbb06 241 1513 1605
sbb04 in KA 9 10 sbb04 1788 3060 1605
Bjh2245 in KA 11 12 Bjh2245 97 1369 1605
sbb16 in CK 13 14 sbb16 90 1290 1473
Bjh1858 in CK 15 16 Bjh1858 33 1233 1473
Bjh1906 in KA 17 18 Bjh1906 47 1319 1605
sbb19 in KA 19 20 sbb19 597 1869 1605
sbb12 in KA 21 22 sbb12 234 1506 1605
sbb09 in KA 23 24 sbb09 35 1307 1605
sbb10 in KA 25 26 sbb10 1026 2298 1605
sbb42 in KA 27 28 sbb42 91 1363 1605
Bjh2294 in KA 29 30 Bjh2294 2507 3779 1605
sbb07 in KA 31 32 sbb07 1431 2703 1605
sbb18 in AK 33 34 sbb18 399 1599 1680
sbb17 in AK 35 36 sbb17 399 1599 1680
sbb12 in AK 37 38 sbb12 234 1434 1680
sbb09 in AK 39 40 sbb09 35 1235 1680
sbb42 in AK 41 42 sbb42 91 1291 1680
Bjh2294 in AK 43 44 Bjh2294 2507 3707 1680
sbb06 in AK 45 46 sbb06 241 1441 1680
sbb20 in AK 47 48 sbb20 597 1797 1680
  • Extra Righties:
    • Stage 0 mehtylated RE map part 3.jpg
    • Layout:
' Part A Lane # Part B Lane # Part Name Part size Eco/xho Frags eco/Xho Frags
sbb08 in KC 1 2 sbb08 35 1101 1605
sbb11 in KC 3 4 sbb11 232 1298 1605
sbb05 in KC 5 6 sbb05 319 1385 1605
Bjh1858 in CA 7 8 Bjh1858 33 1305 1473

Christoph Neyer 20:18, 2 July 2010 (EDT)

Decided to redo all the methylations.

Christoph Neyer 18:40, 29 June 2010 (EDT)

Restriction digest of iGEM10_76 (clones 2-6). Expect bands at ~3100 and ~1700:
Re map of igem 76.jpg

Christoph Neyer 19:03, 28 June 2010 (EDT)

Colony PCR of manually assembled parts iGEM10_076-79:
Colony PCR manual assembly 76-79.jpg

Christoph Neyer 17:45, 28 June 2010 (EDT)

Restriction map of catchup methylated parts:
Restriction map of catchup methylated parts.jpg
Layout:

Part Strain Clone number Part number ' Lane Number Part size eco/xho frags eco/xho frags
8 with 1 R 4 1 1 35 1307 1473
9 with 3 R 4 3 2 33 1099 1605
8 with 1 R 3 1 3 35 1307 1473
9 with 3 R 3 3 4 33 1099 1605
8 with 1 R 2 1 5 35 1307 1473
9 with 3 R 2 3 6 33 1099 1605
8 with 1 R 1 1 7 35 1307 1473
9 with 3 R 1 3 8 33 1099 1605
-- -- --
9 with 23 L 4 23 9 91 1157 1605
5 with 20 L 4 20 10 823 2023 1473
9 with 23 L 3 23 11 91 1157 1605
5 with 20 L 3 20 12 823 2023 1473
9 with 23 L 2 23 13 91 1157 1605
5 with 20 L 2 20 14 823 2023 1473
9 with 23 L 1 23 15 91 1157 1605
5 with 20 L 1 20 16 823 2023 1473
Ladder --
9 with 11 L 4 11 17 46 1112 1605
8 with 21 R B1 21 18 232 1504 1473
9 with 11 L 3 11 19 46 1112 1605
8 with 21 R B2 21 20 232 1504 1473
9 with 11 L 2 11 21 46 1112 1605
8 with 21 R B3 21 22 232 1504 1473
9 with 11 L 1 11 23 46 1112 1605
8 with 21 R B4 21 24 232 1504 1473
-- -- --
9 with 20 L 4 20 25 823 1889 1605
8 with 21 R A1 21 26 232 1504 1473
9 with 20 L 3 20 27 823 1889 1605
8 with 21 R A2 21 28 232 1504 1473
9 with 20 L 2 20 29 823 1889 1605
8 with 21 R A3 21 30 232 1504 1473
9 with 20 L 1 20 31 823 1889 1605
8 with 21 R A4 21 32 232 1504 1473

Christoph Neyer 19:41, 25 June 2010 (EDT)

Made methylated basic parts and plated. Need to take out of incubator on Saturday.
Did manual 2ab test run for:

  • iGEM10_076
  • iGEM10_077
  • iGEM10_078
  • iGEM10_079

Plated on a strip today with four different volumes (20,30,40,50ul).

TO DO:

  • Need to double check 7 w/ 10 Lefty from plate A. Looked like it didn't get digested.
  • Need to colony PCR basic parts and composite parts on Monday and then minprep and restriction map for assembly on Tuesday.

Christoph Neyer 19:41, 25 June 2010 (EDT)

Made methylated basic parts and plated. Need to take out of incubator on Saturday.
Did manual 2ab test run for:

  • iGEM10_076
  • iGEM10_077
  • iGEM10_078
  • iGEM10_079

Plated on a strip today with four different volumes (20,30,40,50ul).

TO DO:

  • Need to double check 7 w/ 10 Lefty from plate A. Looked like it didn't get digested.
  • Need to colony PCR basic parts and composite parts on Monday and then minprep and restriction map for assembly on Tuesday.

Christoph Neyer 21:13, 24 June 2010 (EDT)

TO DO:

  • Make methylated basic parts:
    • 9 with 11
    • 5 with 20
    • 9 with 20
    • 9 with 23
    • 8 with 1
    • 8 with 21
    • 9 with 3
  • Manual 2ab test run for two junctions (4 basic parts -> 2 composite parts):
    • iGEM10_065
    • iGEM10_071
  • Create methylated plate layout and transfer parts to it.
  • Once methylated plate is made restriction map it and sequence a few.

Christoph Neyer 17:54, 24 June 2010 (EDT)

Restricton map of methylated parts from plate A:
Restriction map of methylated parts from plate A.jpg
Layout of parts from plate A:

Parts from plate A ' ' ' ' '
Plasmid Part number Part Size Eco/Xho Frags Eco/Xho Frags Lane #
5 with 20 20 823 2023 1473 1
5 with 25 25 41 1241 1473 2
9 with 11 11 46 1112 1605 3
9 with 20 20 823 1889 1605 4
9 with 23 23 91 1157 1605 5
7 with 10 10 100 1300 1680 6
5 with 15 15 90 1290 1473 7
5 with 3 3 33 1233 1473 8
8 with 1 1 35 1307 1473 9
8 with 21 21 232 1504 1473 10
4 with 10 10 100 1166 1679 11
4 with 18 18 1818 2884 1679 12
4 with 9 9 597 1663 1679 13
9 with 3 3 33 1099 1605 14
Ladder
6 with 11 11 46 1318 1605 15
7 with 13 13 399 1599 1680 16
6 with 12 12 597 1869 1605 17
7 with 14 14 399 1599 1680 18
6 with 19 19 228 1500 1605 19
7 with 19 19 228 1428 1680 20
6 with 2 2 35 1307 1605 21
7 with 2 2 35 1235 1680 22
6 with 22 22 1026 2298 1605 23
7 with 23 23 91 1291 1680 24
6 with 23 23 91 1363 1605 25
7 with 24 24 2400 3600 1680 26
6 with 24 24 2400 3672 1605 27
7 with 5 5 241 1441 1680 28
6 with 4 4 1431 2703 1605 29
7 with 9 9 597 1797 1680 30

Col PCR map of basic parts:
ColPCR basic parts 6-24-10 12 mins.jpg
Layout of ColPCR:

Plasmid Part number Part Size Eco/Xho Frags Eco/Xho Frags Lane #
5 with Bca1091 Bca1091 60 1260 1473 1
6 with Bjh2245 Bjh2245 97 1369 1605 2
6 with 12 12 597 1869 1605 3
5 with 25 25 41 1241 1473 4
9 with 11 11 46 1112 1605 5
9 with 23 23 91 1157 1605 6

Christoph Neyer 20:41, 23 June 2010 (EDT)

Restriction map of methylated parts Plate B:
Part 1:
Re map methylated plate A part 1.jpg
Part 2:
Re map methylated plate A part 2.jpg
Layout:

' RE mapping of ALL methylated parts, Plate B RE mapping plate layout: ' ' '
Ladder
4 with 16 16 90 1156 1679 1
5 with 20 20 823 2023 1473 2
4 with 17 17 1845 2911 1679 3
5 with 23 23 91 1291 1473 4
4 with Bjh1882 Bjh1882 681 1747 1679 5
5 with 25 25 41 1241 1473 6
9 with 11 11 46 1112 1605 7
5 with 3 3 33 1233 1473 8
9 with 13 13 399 1465 1605 9
5 with 8 8 1244 2444 1473 10
9 with 20 20 823 1889 1605 11
5 with Bca1091 Bca1091 60 1260 1473 12
9 with 23 23 91 1157 1605 13
8 with 17 17 1845 3117 1473 14
9 with 8 8 1244 2310 1605 15
8 with 18 18 1818 3090 1473 16
Ladder
6 with 12 12 597 1869 1605 17
7 with 13 13 399 1599 1680 18
6 with 13 13 399 1671 1605 19
7 with 14 14 399 1599 1680 20
6 with 22 22 1026 2298 1605 21
7 with 19 19 228 1428 1680 22
6 with 23 23 91 1363 1605 23
7 with 2 2 35 1235 1680 24
6 with 7 7 1788 3060 1605 25
7 with 23 23 91 1291 1680 26
7 with 10 10 100 1300 1680 27
7 with 24 24 2400 3600 1680 28
7 with 11 11 46 1246 1680 29
7 with 5 5 241 1441 1680 30
8 with 4 4 1431 2703 1473 31
7 with 9 9 597 1797 1680 32
Ladder
6 with 11 11 46 1318 1605 33
5 with 15 15 90 1290 1473 34
6 with 12 12 597 1869 1605 35
5 with 3 3 33 1233 1473 36
6 with 19 19 228 1500 1605 37
8 with 1 1 35 1307 1473 38
6 with 2 2 35 1307 1605 39
8 with 21 21 232 1504 1473 40
6 with 22 22 1026 2298 1605 41
8 with 6 6 319 1591 1473 42
6 with 23 23 91 1363 1605 43
6 with 5 5 241 1513 1605 44
6 with 24 24 2400 3672 1605 45
6 with 7 7 1788 3060 1605 46
6 with 4 4 1431 2703 1605 47
6 with Bjh2245 15 90 1362 1605 48
Ladder
Gel #2
Ladder
4 with 10 10 100 1166 1679 1
4 with 18 3 33 1099 1679 2
4 with 9 18 1818 2884 1679 3
9 with 3 1 35 1101 1605 4
6 with 24 24 2400 3672 1605 5
7 with 23 23 91 1291 1680 6
7 with 24 24 2400 3600 1680 7
9 with 11 11 46 1112 1605 8
9 with 23 23 91 1157 1605 9
5 with Bca1091 Bca1091 60 1260 1473 10
4 with Bjh1882 Bjh1882 681 1747 1679 11
6 with Bjh2245 Bjh2245 97 1369 1605 12
6 with 12 12 597 1869 1605 13
5 with 25 25 41 1241 1473 14


Gel #2:
Re map methylated plate A part 3 and unmethylated parts col 9 and 10.jpg

Christoph Neyer 17:44, 23 June 2010 (EDT)

Restriction map of unmethylated parts:
RE map unmeth.JPG

Christoph Neyer 13:06, 23 June 2010 (EDT)

Restriction Map of methylated parts plate rows A and E:
Restriction map of methylated parts rows A and E.jpg
Layout:

' Part number Part Size Eco/Xho Frags Eco/Xho Frags Lane #
4 with 16 16 90 1066 1769 1
9 with 13 13 399 1066 2004 2
5 with 20 20 823 1200 2296 3
5 with 8 8 1244 1200 2717 4
6 with 12 12 597 1272 2202 5
6 with 7 7 1788 1272 3393 6
7 with 13 13 399 1200 2079 7
7 with 23 23 91 1200 1771 8
6 with 11 11 46 1272 1651 9
6 with 22 22 1026 1272 2631 10
5 with 15 15 90 1200 1563 11
8 with 6 6 319 1272 1792 12
4 with 10 10 100 1066 1779 13

Christoph Neyer 13:16, 22 June 2010 (EDT)

Restriction Map Rerun:
Stage1 Restriction Map rerun 6-22.jpg
Layout:

Restriction Mapping ' Lane #s A B Part size eco/xho frags eco/xho frags
9 with iGEM10_023 R 1 2 1879 2945 1605
9 with iGEM10_024 R 3 4 1522 2588 1605
9 with iGEM10_028 L 5 6 157 1223 1605
9 with iGEM10_025 R 7 8 323 1389 1605
9 with iGEM10_027 L 9 10 858 1924 1605
8 with iGEM10_029 L 11 12 1909 3181 1473
9 with iGEM10_034 R2 13 14 1064 2130 1605
5 with iGEM10_035 R 15 16 996 2196 1473
7 with iGEM10_040 R 17 18 771 1971 1680
9 with iGEM10_008 R 19 20 1057 2123 1605
6 with iGEM10_033 L 21 22 1464 2736 1605

Daniela Mehech 14:19, 21 June 2010 (EDT)

Colony PCR results from Group 2
ColonyPCRredoneparts.JPG
lane 1,3,5,7 = 5 w/ 35 L
lane 2,4,6,8 = 5 w/ 38
lane 9,11,13,15 = 5 w/ 35 R
lane 10,12,14,16 = 6 w/33 L
lane 17,19,21,23 = 6 w/33 R
lane 18,20,22,24 = 9 w/ 8 L
lane 25, 27,29,31 = 7 w/40
lane 26,28,30,32 = 9 w/ 34 L
lane 33,34,35,36 = 9 w/ 8 R
lane 37,38,39,40 = 9 w/ 34 R
Except for first 8 lanes, everything looked good and we mini-prepped two of each. Mini-prepped lanes 1-8 too and will send them for sequencing


Colony PCR results for group 3:
ColonyPCRforgottenparts.JPG
Loaded as: 1,9,2,10,3,11,4,12,5,13,6,14,7,15,8,16,17,25,18,26,19,27,20,28,21,29,22,30,23,31,24,32

Colony PCR results for group 4:
ColonyPCRrepickedcolonies.JPG

Restriction Mapping of everything we've mini-prepped (groups 1 and 2)
REmappingStage1Round1RightHalf.JPG
Right Half
REmappingStage1Round1LeftHalf.JPG
Left Half

To Do

Check for co-transformation for Groups 3 and 4
Mini-Prep good colonies of group 3 and 4
Send to sequencing some parts?
Begin Stage 2 (hopefully all our stage 1 parts are right)

Daniela Mehech 21:28, 18 June 2010 (EDT)

We labeled our transformation plate wrong so when we went to plate the cells many of them were plated on the wrong antibiotics. We still had colonies grow because they were co-transformed and had resistance to all antibiotics. This also explains why so many colonies grew when we tested for co-transformation. We have split our stage 1 parts into 4 groups:

1. Colonies grew on proper antibiotics and were not co-transformed. There are 12 of these. We mini-prepped these today and will do restriction mapping properly on Monday
2. Colonies that grew on wrong antibiotics need to be redone. There are 10 of these.We digested, ligated and transformed them today.
3. Parts that we thought we hadn't done (see yesterday's notes), but it turns out we did do them without realizing it since our plates were labeled wrong. Since we did them twice and they were all plated correctly the second time we will pick colonies, do Colony PCR, check for co-transformation and mini-prep these 9 on Monday.
4. Colonies that grew on the right antibiotics but were co-transformed. We will pick more colonies from the original plate to do ColPCR

Daniela Mehech 18:37, 17 June 2010 (EDT)

We got cell growth for every part we plated (although in some parts there were very few colonies, about 3 or 4).
We made a Colony PCR Mastermix and tried to distribute it to two PCR plates with the robot but the robot kept giving us an error message (It said it couldn't pipette 19uL if there was 17uL in the tube but there was 2 ml in the tube and we wanted it to pipette 17uL). We gave up and I pipetted all 136 wells by hand.
Christoph and I picked colonies, added them to the PCR plate, innoculated them in a 96-well plate and left them in the incubator. We ran a ColPCR3K program on the both PCR plates.
Christoph noticed that we had based all of our csv robot files from the wrong list. We made all of the parts we needed but some of the parts had to be inserted into multiple vector backbones. We began making the 6 forgotten parts by hand. 3 of these parts need to be transformed into both Righties and Lefties so we're making 9 parts by hand.
We dabbed broth from our 96-well inoculated plate on both AKC plates and LB-only plates as negative and positive controls respectively for whether we picked any co-transformed colonies.
We need to run a huge gel when the PCR finishes at 4:40pm. Then we need to analyze the results. Christoph already named all the new composite parts and calculated their sizes.

To Do

We will mini-prep our good colonies tomorrow. Hopefully we'll have at least two good copies for each part so that we can continue with Plates A and B for stage 2.
We can start planning for Stage 2 assembly when we have free time. If we do it beforehand hopefully we'll catch any mistakes before running the robot
We should make a list of all the information Clothos gives us after it solves for the assembly tree. We've had to spent a good chunk of time writing excel formulas and making spreadsheets just so we know exactly how to assemble everything

Christoph Neyer 21:26, 18 June 2010 (EDT)

Did Eco/Bam digest of good parts and here is the layout on the gel:
After about 20mins:
Eco-Bam digest map stage 1 part set 1 time A.jpg Eco-Bam digest map stage 1 part set 1 time B.jpg

' ' ' ' ' Lane # '
part size +200 Eco/Bam frags Eco/Bam frags A B
7 with iGEM10_022 R 2078 4755 4755 1 2
9 with iGEM10_023 R 2079 4624 4624 3 4
9 with iGEM10_024 R 1722 4267 4267 5 6
9 with iGEM10_028 L 357 157 2745 7 8
4 with iGEM10_036 R 282 2827 2827 9 10
4 with iGEM10_037 R 566 3111 3111 11 12
4 with iGEM10_009 R 479 3024 3024 13 14
9 with iGEM10_025 R 523 3068 3068 15 16
9 with iGEM10_027 L 1058 858 2745 17 18
8 with iGEM10_029 L 2109 1909 2671 19 20
9 with iGEM10_026 L 1535 1335 2745 21 22
9 with iGEM10_027 R 1058 3603 3603 23 24

Christoph Neyer 15:51, 18 June 2010 (EDT)

Found out that we mixed up some things while creating the plate layouts for stage1 and we ended up plating on the wrong antibiotics.

  • We need to spot check and colony PCR the catchups we started yesterday. (9 total):
Name Strain Lefty Input Righty Input
5 with iGEM10_026 L 9 with 8 7 with 23
5 with iGEM10_027 L 9 with 20 7 with 2
5 with iGEM10_032 L 9 with 23 7 with 13
5 with iGEM10_034 L 9 with 20 7 with 5
5 with iGEM10_008 L 9 with 20 7 with 19
8 with iGEM10_035 L 6 with 13 4 with 9
5 with iGEM10_034 R 9 with 20 7 with 5
5 with iGEM10_008 R 9 with 20 7 with 19
8 with iGEM10_035 R 6 with 13 4 with 9
  • Assembled these parts that we missed/plated on the wrong antibiotics manually:
Name Strain Lefty Input Righty Input
5 with iGEM10_035 L 9 with 13 7 with 9
6 with iGEM10_033 L 8 with 4 9 with 3
9 with iGEM10_034 L 5 with 20 6 with 5
9 with iGEM10_008 L 5 with 20 6 with 19
5 with iGEM10_035 L 9 with 13 7 with 9
5 with iGEM10_038 R 9 with 11 7 with 24
7 with iGEM10_040 R 4 with Bjh1882 5 with 15
6 with iGEM10_033 R 8 with 4 9 with 3
9 with iGEM10_034 R 5 with 20 6 with 5
9 with iGEM10_008 R 5 with 20 6 with 19
5 with iGEM10_035 R 9 with 13 7 with 9

Christoph Neyer 13:30, 18 June 2010 (EDT)

We spot checked our colony PCR colonies on ACK to check for co-transformation and this is what our plates look like:
Plate A
Colony Co-transformation Spot Check Stage1A.jpg

and Plate B
Colony Co-transformation Spot Check Stage1B.jpg

Christoph Neyer 21:22, 17 June 2010 (EDT)

Colony PCR results for Stage1 parts:
Here are columns 1-6 in two parts:
Part 1 (Parts 1 and 2 are picutres of the same gels)
Colony PCR Stage1 A+B Columns 1-6 part 1.jpg
and Part 2
Colony PCR Stage1 A+B Columns 1-6 part 2.jpg
Here are columns 7-10:
Colony PCR Stage1 A+B Columns 7-10.jpg

Christoph Neyer 18:24, 17 June 2010 (EDT)

Most of the colonies turned out good for Stage 1 of the robot assembly. We picked colonies for colony PCR.

Manually made these parts for Stage 1 using the robot protocol. We missed them some how:

Name Strain Lefty Input Righty Input
5 with iGEM10_026 L 9 with 8 7 with 23
5 with iGEM10_027 L 9 with 20 7 with 2
5 with iGEM10_032 L 9 with 23 7 with 13
5 with iGEM10_034 L 9 with 20 7 with 5
5 with iGEM10_008 L 9 with 20 7 with 19
8 with iGEM10_035 L 6 with 13 4 with 9
5 with iGEM10_034 R 9 with 20 7 with 5
5 with iGEM10_008 R 9 with 20 7 with 19
8 with iGEM10_035 R 6 with 13 4 with 9

Christoph Neyer 21:15, 16 June 2010 (EDT)

While plating Stage1 2ab assembly set A we may have reversed the order of Col 1 B (30 ul).
The cells from plate A3-D3 may be in wells B4-B1 instead of B1-B4 on the 2x12 plated for Col 1A from the reaction plate.
Also note rows A and B were switched on the plate for Col 5A as noted on the plate. A is 30ul and B is 60ul instead of the other way around.

Christoph Neyer 18:10, 16 June 2010 (EDT)

Redoing Stage1B and doing Stage1A 2ab assembly today.
Check autoclave at 4:30.
Decided to redo all of B along with A. We can do them together on the robot by doubling everything but changing the source plate Made dilution plate A, and added 60uL of dilution to dilution plate B. 7 with 11 still looked different Distributed digestion cocktail to both dilution plates Note: Robot begins pipetting air if it uses the same tip for two long. We stopped the program halfway through to change its tip Distributed Righty and Lefty parts to reaction plates. Made sure there were duplicate columns for parts being transformed twice Checked that at this point all wells had the same volume Put plates in thermocycler to incubate and heat kill Distributed antibiotics to 10 plates. All of LB agar was contaminated so will pour that during the rescue step added ligation mixture to each well and let incubate at room temperature.

Christoph Neyer 18:11, 16 June 2010 (EDT)

Robot 2AB assembly Stage 1

Yesterday we did stage 1 of our 2AB assembly only on plate B. Today we will repeat the process on plate A without making all of the mistakes that we did yesterday.

June 15:
Autoclaved our strips for plating. Learned how to work the autoclave machine

Made a new plate layout for the reaction plate. Wells are organized by what strain the part needs to be transformed into to
MISTAKE: Did not listen to Tim and organized wells by column instead of rows. Rows are better because the plating strips have 2 rows and 12 columns
MISTAKE: Did not read procedure all the way through in detail and assumed that we could use a well for two transformations. Our dilution and reaction plate needs to have duplicates of the parts that go into both Righty and Lefty strains. We have to add the duplicates in either the dilution or reaction plate
Made the dilution plate B (30ul of DNA into each well + 30ul of NEB2 stock diluted by a factor of 5). Dilution plate had the same layout as methylated stock plate so we did it by hand. Next time, we should consider adding duplicates of the parts being transformed twice.
NOTE: 7 with 11 was frozen in our methylated stocks plate. This is the part that was mini-prepped separately the day before. All other wells weren't frozen.

Had the robot make reaction plate (distribute digestion mastermix to all wells, distribute all Lefty parts, mix, distribute all righty parts, mix)
While the reaction plate incubated in the black thermocycler (which also did heat kill of the digestion enzymes) we distributed the antibiotics to the 24-well strips. We made antibiotics (dilute stock by 10, add 1ul of dye). This part went fine except we should have labeled the plates before we put them on the robot. Added LB agar to the plates and let them dry next to the flame.
MISTAKE: forgot to make second plate for parts being transformed twice. Column 5 in reaction plate needed to go to two different plates. We made the extra plate by hand

Had robot add the ligation cocktail to the wells and let it incubate at room temperature. We began thawing our bacteria.
Did transformation of bacteria. We had enough volume to seed at least 60uL of bacteria per column.
MISTAKE: Did not realize that the protocol was for seeding 10uL of bacteria. We wanted to seed about 150uL. We should not have done the centrifugation and aspiration of the supernatant step.
MISTAKE: ejecting tips from multi-channel before releasing cells on plate. We lost 60uL of our cells from column 7.
MISTAKE: We split the volume in column 5 into column 9 (because column 5 had to be transformed into both righty and lefty strains). Thus we also did not have enough volume for those parts.
MISTAKE: not taking a short break after lunch to re-energize and prevent making silly mistakes

Christoph Neyer 20:10, 15 June 2010 (EDT)

  • Reorganize destination plate based on L and R not on antibiotics. CHECK
  • AUTOCLAVE 24 well Strips! CHECK
  • Digesting (1hr) Check
    • Dilute NEB2 and DNA.
  • Make plates (2hr) Check
    • Cherry pick antibiotics Check
  • Heat kill (20 mins)Check
  • Ligation (30 mins)Check
  • Transform + Rescuing (1hr) Check
  • Plate (1hr)Check

Forgot to make two copies of the parts that are both lefty and righty. So, we just used half of ligation product of each.
Here is the layout of the transformation plate:
Stage1Btransformationlayout.png
Column 5 is Lefty and was transformed with only half the ligation product.
Column 9 is righty and was also transformed with half the ligation product.

Christoph Neyer 21:29, 14 June 2010 (EDT)

Goal for tomorrow is to transform and plate stage 1A and 1B. That involves:

  • Reorganize destination plate based on L and R not on antibiotics.
  • AUTOCLAVE 24 well Strips!
  • Digesting (1hr)
    • Dilute NEB2 and DNA.
  • Make plates (2hr)
    • Cherry pick antibiotics
  • Heat kill (20 mins)
  • Ligation (30 mins)
  • Transform + Rescuing (1hr)
  • Plate (1hr)

Christoph Neyer 14:18, 14 June 2010 (EDT)

Thanks to Tim and Shelly for picking colonies from methylation transformation

  • May have mislabeled these tubes, so we are running a BglII/Xho map:

Put in digest at 11am. Run gel at 11:30.
BgXh mapping stage 0 June 14.JPG
Looks ok. Added these parts to methylated parts plates

Lane # ID Name Expected fragments '
1 4 with 10 #1 pMLL4-AC+B10sbb43 850 1990
2 4 with 10 #2 pMLL4-AC+B10sbb43 850 1990
3 4 with 18 #1 pMLL4-AC+B10sbb13 850 2160
4 4 with 18 #2 pMLL4-AC+B10sbb13 850 2160
5 4 with 9 #1 pMLL4-AC+B10Sbb20 850 2450
6 4 with 9 #2 pMLL4-AC+B10Sbb20 850 2450
7 9 with 3 #1 pMLL9-CA+Bjh1853 300 1500
8 9 with 3 #2 pMLL9-CA+Bjh1853 300 1500

Christoph Neyer 21:36, 11 June 2010 (EDT)

Plated Transformations from robot dilution.

Christoph Neyer 19:04, 11 June 2010 (EDT)

Next step is to transform each part into a Righty or Lefty strain.
Manually transformed parts from dilution plate into Righty and Lefty strains. Incubated at 5:10. Take out at 5:50.
Transformation plate has same layout as dilution plate below. Here is layout again:

Transformation plate layout

Columns 1-6 are in Righty strains. Columns 7-12 are in Lefty strains.

' 1 2 3 4 5 6 7 8 9 10 11 12
A 4 with 16 5 with 20 6 with 12 7 with 10 8 with 17 9 with 11 4 with 10 6 with 11 6 with 5 7 with 13 8 with 1 9 with 3
B 4 with 17 5 with 23 6 with 13 7 with 11 8 with 18 9 with 13 4 with 18 6 with 12 6 with 7 7 with 14 8 with 21
C 4 with Bjh1882 5 with 25 6 with 22 8 with 4 9 with 20 4 with 9 6 with 19 6 with Bjh2245 7 with 19 8 with 6
D 5 with 3 6 with 23 9 with 23 6 with 2 7 with 2
E 5 with 8 6 with 7 9 with 8 6 with 22 7 with 23
F 6 with Bca1091 6 with 23 7 with 24
G 5 with 15 6 with 24 7 with 5
H 5 with 3 6 with 4 7 with 9

Christoph Neyer 16:31, 11 June 2010 (EDT)

Did a test run with the robot using our dilution csv file. Checked cloumn 4 it worked well.
Miniprepped pMLL5+Bca1152 and sent in for sequencing as iGem021.
Using robot to dilute 4ul of each vector+part stage 0 assembly into 76ul of water. See this csv file: Media:Stage0PartsDilution.csv

Source plate layout:

' 1 2 3 4 5 6 7 8 9 10 11 12
A 6+2 6+11 6+22 7+9 7+24 5+23 8+17 4+10 9+3 9+20 7+24
B 6+2 6+11 6+22 7+10 5+3 5+23 8+17 4+10 9+3 9+20 9+11
C 6+4 6+12 6+23 7+11 5+3 8+1 8+18 4+16 9+8 9+23 9+23
D 6+4 6+12 6+23 7+13 5+8 8+1 8+18 4+16 9+8 9+23 pMLL5+Bca1091 (L)
E 6+5 6+13 6+24 7+14 5+8 8+4 8+21 4+17 9+11 pMLL4+Bjh1882 (L)
F 6+5 6+13 6+24 7+19 5+15 8+4 8+21 4+17 9+11 pMLL6+Bjh2245
G 6+7 6+19 7+2 7+23 5+20 8+6 4+9 4+18 9+13 6 +24 6+12
H 6+7 6+19 7+5 7+24 5+20 8+6 4+9 4+18 9+13 7 + 23 5+25 (D)

Destination plate layout:

Columns 1-6 need to be transformed into Righty strains. Columns 7-12 need to be transformed into Lefty strains.

' 1 2 3 4 5 6 7 8 9 10 11 12
A 4 with 16 5 with 20 6 with 12 7 with 10 8 with 17 9 with 11 4 with 10 6 with 11 6 with 5 7 with 13 8 with 1 9 with 3
B 4 with 17 5 with 23 6 with 13 7 with 11 8 with 18 9 with 13 4 with 18 6 with 12 6 with 7 7 with 14 8 with 21
C 4 with Bjh1882 5 with 25 6 with 22 8 with 4 9 with 20 4 with 9 6 with 19 6 with Bjh2245 7 with 19 8 with 6
D 5 with 3 6 with 23 9 with 23 6 with 2 7 with 2
E 5 with 8 6 with 7 9 with 8 6 with 22 7 with 23
F 6 with Bca1091 6 with 23 7 with 24
G 5 with 15 6 with 24 7 with 5
H 5 with 3 6 with 4 7 with 9

Christoph Neyer 13:57, 11 June 2010 (EDT)

  • Added grown up Lefty and Righty strains to 500ml each of LB. Growing up for 2-3hrs. Check at 1pm.
  • Running colony PCR gel for Bca1152+pMLL5. Expected band at ~260.

Colony PCR Bca1152.JPG
Colonies A,C,D look good. Chose C and D to miniprep. Cells were put in warm room at 9am.
Grab Bca1152 colony PCR colonies at 1 or 2pm.

  • Added missing parts to iGEM10 plate1 minis:
' 1 2 3 4 5 6 7 8 9 10 11 12
A 6+2 6+11 6+22 7+9 7+24 5+23 8+17 4+10 9+3 9+20 7+24 Lysis Device
B 6+2 6+11 6+22 7+10 5+3 5+23 8+17 4+10 9+3 9+20 9+11 5+25
C 6+4 6+12 6+23 7+11 5+3 8+1 8+18 4+16 9+8 9+23 9+23
D 6+4 6+12 6+23 7+13 5+8 8+1 8+18 4+16 9+8 9+23 pMLL5+Bca1091 (L)
E 6+5 6+13 6+24 7+14 5+8 8+4 8+21 4+17 9+11 pMLL4+Bjh1882 (L)
F 6+5 6+13 6+24 7+19 5+15 8+4 8+21 4+17 9+11 pMLL6+Bjh2245
G 6+7 6+19 7+2 7+23 5+20 8+6 4+9 4+18 9+13 6 +24 6+12
H 6+7 6+19 7+5 7+24 5+20 8+6 4+9 4+18 9+13 7 + 23

Christoph Neyer 21:03, 10 June 2010 (EDT)

TO DO:

  • Pick colonies from Bca1152 and do colony PCR. Then miniprep if time.
  • Play with robot
  • Make competent cells.
  • Transfer box parts to dilutions plate.
  • When oligos arrive. Attempt biobrick on Bjh2342CA.

Christoph Neyer 13:38, 10 June 2010 (EDT)

  • Had to re-grow Lefty and Righty strains since no colonies grew.
  • Need to make part 5 with 25 (pMLL5-CK+B10sbb37) or (pMLL5-CK+Bca1152)

Currently in DH10B. Need to Eco/Bam transfer it into pMLL5-CK.
Put E/B digest of Bca1152 in DH10B into thermocycler at 12:10pm. Take out at 1:10pm and do small fragment zymo cleanup.
Set up ligation of Bca1152 and pMLL5 at 2:15. Transform at 2:45.
Heat-shock transformed Bca1152+pMLL5 into Jtk049. Plate at 4:15pm.
Plated Bca1152+pMLL5 into Jtk049. Growing up over night.
Sent in iGEM020 for sequencing(6 with 12C from working box to double check).

Christoph Neyer 20:31, 9 June 2010 (EDT)

TO DO List:

  • Finish generating competent cells for "Lefty" and "Righty" strains.
  • Analyze sequencing data for pMLL6+Bjh2245 B and C.
  • Transform pMLL6+Bjh2245 into "Righty" strain.
  • Transform other parts into the correct "Righty" or "Lefty" strains to prepare for robot 2ab assembly.

Christoph Neyer 15:16, 9 June 2010 (EDT)

Colony PCR Bjh2245.JPG
Colony PCR of pMLL6+Bjh2245 should be at about 265bps. Looks good. Wait for colonies to grow and then miniprep.

  • Sent in miniprepped pMLL4-Bjh1882 (D) and pMLL5-Bca1091 (2A) for sequencing.
  • Started growing up Lefty and Righty strains. Picked two colonies of each lefty and righty and grew them up in 10ml LB.
  • Designed three potential oligos for biobricking part Bjh2341CA (igemTen011, igemTen012, and igemTen013)
  • Miniprepped pMLL6+Bjh2245. Need to transform into "Righty strain"
  • Sent in minprepped pMLL6+Bjh2245 to be sequenced.

Christoph Neyer 13:28, 9 June 2010 (EDT)

Bca1091 is 60bps.
Colony PCR band for parts:

  • pMLL4+bjh1882 = 849bps
  • pMLL6+bjh2245 = 265bps
  • pMLL5+bca1091 = 228bps
  • For First try Colony PCR of 1882 and 1091:
    • Bca1091 pick lanes 8 and 10 for miniprepping.(Block #1: Colonies from well 1D and 2A)


  • For Second try Colony PCR of 1882 and 1091
    • Bjh1882 pick lanes 1 and 4 for miniprepping. (Block #2: Colonies from well A and D)

Christoph Neyer 19:37, 8 June 2010 (EDT)

TO DO Tomorrow:

  • Miniprep colonies that we pick for pMLL4+Bjh1882 and pMLL5+Bca1091. These are methylated correctly for the robot.
  • Colony PCR pMLL6+Bjh2245 in Jtk049. These are not methylated correctly.

Christoph Neyer 19:51, 8 June 2010 (EDT)

  • Ran gel of colony PCR pMLL4-Bjh1882 and pMll5-Bca1091 AGAIN:

Colony PCR 2245+1091 Second try.JPG

ladder 1882a b c d 1901a(1) b c d ladder 1901a(2) b c d

Christoph Neyer 16:59, 8 June 2010 (EDT)

  • Ligated pMLL6 Eco/Bam digest with part Bjh2245.
  • Ran gel of colony PCR pMLL4-Bjh1882 and pMll5-Bca1091:

Colony PCR 2245+1091 First try.JPG

ladder 1882a b c d 1901a(1) b c d ladder 1901a(2) b c d
  • Repicking colonies since Colony PCR appeared contaminated. Block has same layout, shown below:
PCR Block 1882 and 1091 #2 ' ' ' '
1882 1091 #1 1091 #2
A A A
B B B
C C C
D D D
  • Ran out of "Righty" cells. So we are transforming Jtk049 with our E/B ligation of pMLL6+Bjh2245.

Christoph Neyer 14:04, 8 June 2010 (EDT)

  • Digested pMLL6 w/ Eco/Bam and gel purified.
  • Colony PCR of transformed pMLL4-Bjh1882 and pMLL5-Bca1091:
PCR Block 1882 and 1091 #1 ' ' ' '
1882 1091 #1 1091 #2
A A A
B B B
C C C
D D D

Christoph Neyer 19:30, 7 June 2010 (EDT)

Bjh1882 Vectors: AC
Bjh2245 Vectors: KA
Bca1091 Vectors: CK
pMLL4-AC
pMLL5-CK
pMLL6-KA
pMLL7-AK
pMLL8-KC
pMLL9-CA
Bjh1882+pMLL4-AC = Lefty
Bjh2245+pMLL6-KA = Righty
Bca1091+pMLL5-CK = Lefty
Ligated isolated Bjh1882 and Bca1091 into above vectors. Transformed into Lefty strains.
Need to ligate Bjh2245 (Missing pMLL6, need to digest).
TO DO:

  • Digest pMLL6 with Eco/Bam and gel purify. Then ligate with Bjh2245.
  • Check transformed Bjh1882 and Bca1091.
  • Transform ligated Bjh2245+pMLL6 into Righty strain

Christoph Neyer 16:35, 7 June 2010 (EDT)

Couldn't see fragments. Turns out Bjh2245 is 97bp and Bjh1882 is ~680bps.
Re-cut Bjh2245 and Bjh1882.
Small fragment zymo cleanup of Bjh2245 digest.

Gel purify bjh1882.JPG
Gel purified Bjh1882.

Christoph Neyer 15:54, 7 June 2010 (EDT)

Eco/Bam digest of parts: Bjh2245 (LifeACT), Bjh1882 (RFP), and BCA1091 (Ptet).
Gel purified Bjh1882 and Bjh2245.
Small fragment zymo cleanup of BCA1091.