Berk2010-Christoph

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(Christoph Neyer 14:52, 28 July 2010 (EDT))
(Christoph Neyer 14:52, 28 July 2010 (EDT))
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*Assemble the rest of the stage 4 parts
*Assemble the rest of the stage 4 parts
 +
==[[User:Christoph Neyer|Christoph Neyer]] 18:42, 30 July 2010 (EDT)==
 +
Stage 4 repicks from Samwise:
 +
Gels: <br>
 +
Part A: <br>
 +
[[Image:Stage 4 Round 1 repicks from Samwise.jpg]]<br>
 +
Part B: <br>
 +
[[Image:Stage 4 Round 1 repicks from Samwise Part B.jpg]]<br>
 +
Layout: <br>
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|'''Stage 4 Round 1 Re-picks'''
 +
| align="center" style="background:#f0f0f0;"|'''Lanes'''
 +
| align="center" style="background:#f0f0f0;"|''''''
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| align="center" style="background:#f0f0f0;"|'''Part Name'''
 +
| align="center" style="background:#f0f0f0;"|'''Part size'''
 +
| align="center" style="background:#f0f0f0;"|'''Eco/xho Frags'''
 +
| align="center" style="background:#f0f0f0;"|'''eco/Xho Frags'''
 +
|-
 +
| iGEM10_117 in CA||1,3,5,7,9,11,13,15||||iGEM10_117||6246||7518||1473
 +
|-
 +
| iGEM10_127 in CK||2,4,6,8,10,12,14,16||||iGEM10_127||4187||5387||1473
 +
|-
 +
| iGEM10_129 in CK||17,19,21,23,25,27,29,31||||iGEM10_129||5740||6940||1473
 +
|-
 +
| iGEM10_133 in CA||18,20,22,24,26,28,30,32||||iGEM10_133||3697||4969||1473
 +
|-
 +
| iGEM10_136 in CA||33,35,37,39,41,43,45,47||||iGEM10_136||4096||5368||1473
 +
|-
 +
| iGEM10_138 in CA||34,36,38,40,42,44,46,48||||iGEM10_138||4096||5368||1473
 +
|}
==[[User:Christoph Neyer|Christoph Neyer]] 14:52, 28 July 2010 (EDT)==
==[[User:Christoph Neyer|Christoph Neyer]] 14:52, 28 July 2010 (EDT)==
Assembled Stage 4 Parts and some Stage 3 catchups. <br>
Assembled Stage 4 Parts and some Stage 3 catchups. <br>

Revision as of 18:42, 30 July 2010

Contents

TO DO

  • Assemble the rest of the stage 4 parts

Christoph Neyer 18:42, 30 July 2010 (EDT)

Stage 4 repicks from Samwise: Gels:
Part A:
Image:Stage 4 Round 1 repicks from Samwise.jpg
Part B:
Image:Stage 4 Round 1 repicks from Samwise Part B.jpg
Layout:

Stage 4 Round 1 Re-picks Lanes ' Part Name Part size Eco/xho Frags eco/Xho Frags
iGEM10_117 in CA1,3,5,7,9,11,13,15iGEM10_117624675181473
iGEM10_127 in CK2,4,6,8,10,12,14,16iGEM10_127418753871473
iGEM10_129 in CK17,19,21,23,25,27,29,31iGEM10_129574069401473
iGEM10_133 in CA18,20,22,24,26,28,30,32iGEM10_133369749691473
iGEM10_136 in CA33,35,37,39,41,43,45,47iGEM10_136409653681473
iGEM10_138 in CA34,36,38,40,42,44,46,48iGEM10_138409653681473

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:

Longer run time to better separate bands:

Layout:

' ' ' ' ' ' '
Clones A and B are Gel #1Clones C and D are on Gel #2
Stage 4 parts + Stage 3 Catchups from PippinLanes (Clones A and C)Lanes (Clones B and D)Part NamePart sizeEco/xho Fragseco/Xho Frags
iGEM10_117 in CA12iGEM10_117624675181473
iGEM10_118 in CA34iGEM10_118413854101473
iGEM10_119 in CA56iGEM10_119664579171473
iGEM10_120 in CA78iGEM10_120413854101473
iGEM10_121 in CA910iGEM10_121664579171473
iGEM10_132 in CA1112iGEM10_132373950111473
iGEM10_133 in CA1314iGEM10_133369749691473
iGEM10_136 in CA1516iGEM10_136409653681473
iGEM10_088 in CA1718iGEM10_088109623681473
iGEM10_088 in CA1920iGEM10_088109623681473
iGEM10_127 in CK2122iGEM10_127418753871473
iGEM10_128 in CK2324iGEM10_128567668761473
iGEM10_129 in CK2526iGEM10_129574069401473
iGEM10_138 in CA2728iGEM10_138409653681473
iGEM10_139 in CA2930iGEM10_139347447461473
iGEM10_103 in KA3132iGEM10_103572569971605
iGEM10_103 in KA3334iGEM10_103572569971605
iGEM10_104 in KA3536iGEM10_104486961411605
iGEM10_110 in KA3738iGEM10_110321844901605
iGEM10_110 in KA3940iGEM10_110321844901605

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:

Layout:

' Clones A and B are Gel #1 Clones C and D are on Gel #2 ' ' ' '
Stage 3 parts + Stage 2 Cathups from GandalfLanes (Clones A and C)Lanes (Clones B and D)Part NamePart sizeEco/xho Fragseco/Xho Frags
iGEM10_105 in CA12iGEM10_105233136031473
iGEM10_107 in CA34iGEM10_107316944411473
iGEM10_109 in CA56iGEM10_109323345051473
iGEM10_088 in AC78iGEM10_088109621621679
iGEM10_084 in KC910iGEM10_084215432201605
iGEM10_101 in KA1112iGEM10_101491161831605
iGEM10_102 in KA1314iGEM10_102240436761605
iGEM10_108 in AK1516iGEM10_108101822181680
iGEM10_111 in KA1718iGEM10_111236236341605
iGEM10_114 in KA1920iGEM10_114174030121605
iGEM10_112 in CA2122iGEM10_112203033021473
iGEM10_113 in CA2324iGEM10_113204933211473
iGEM10_115 in CA2526iGEM10_115196632381473
iGEM10_116 in CA2728iGEM10_116198532571473
iGEM10_085 in CA2930iGEM10_085357148431473
iGEM10_087 in CA3132iGEM10_087356448361473
iGEM10_097 in CA3334iGEM10_097227835501473

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:

Gel Part B:

Small Gel:

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 CA12iGEM10_077187931511473
iGEM10_078 in CA34iGEM10_078152227941473
iGEM10_079 in CA56iGEM10_079111723891473
iGEM10_056 in AC78iGEM10_0568211481679
iGEM10_061 in CA910iGEM10_061106423361473
iGEM10_063 in CA1112iGEM10_063105723291473
iGEM10_066 in CA1314iGEM10_0668013521473
iGEM10_062 in AC1516iGEM10_06227913451679
iGEM10_054 in CA1718iGEM10_054133526071473
iGEM10_067 in CA1920iGEM10_06715714291473
iGEM10_068 in AK2122iGEM10_06877119711680
iGEM10_055 in KA2324iGEM10_055146427361605
iGEM10_055 in KA2526iGEM10_055146427361605
iGEM10_065 in CK2728iGEM10_065255437541473
iGEM10_057 in CK2930iGEM10_05785820581473
LadderLadder
iGEM10_071 in KC3132iGEM10_071190929751605
iGEM10_072 in KC3334iGEM10_07269717631605
iGEM10_061 in CK3536iGEM10_061106422641473
iGEM10_063 in CK3738iGEM10_063105722571473
iGEM10_073 in CK3940iGEM10_07399621961473
iGEM10_074 in CK4142iGEM10_07449016901473
EMPTY4344EMPTY#N/A#N/A#N/A
iGEM10_054 in CK4546iGEM10_054133525351473
Small gel
iGEM10_076 in AK12iGEM10_076187830781680
iGEM10_057 in CA34iGEM10_05785821301473
iGEM10_060 in AC56iGEM10_06036614321679
iGEM10_075 in CK78iGEM10_07549016901473
iGEM10_065 in CK910iGEM10_065255437541473

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

Stage 2 Parts RE map from Frodo:
Gel:

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 NamePart sizeEco/xho Fragseco/Xho Frags
iGEM10_080 in KC12iGEM10_080154626121605
iGEM10_082 in CK34iGEM10_082173429341473
iGEM10_083 in CK56iGEM10_083173429341473
iGEM10_084 in KC78iGEM10_084215432201605
iGEM10_086 in KC910iGEM10_086130523711605
iGEM10_081 in CA1112iGEM10_081336546371473
iGEM10_093 in CA1314iGEM10_093104323151473
iGEM10_094 in CA1516iGEM10_094227835501473
iGEM10_095 in CA1718iGEM10_095195432261473
LadderLadder#N/A#N/A#N/A
iGEM10_096 in CA1920iGEM10_096151627881473
iGEM10_098 in CA2122iGEM10_098195432261473
iGEM10_099 in CA2324iGEM10_099151627881473
iGEM10_100 in CA2526iGEM10_100108723591473
iGEM10_089 in KA2728iGEM10_089192531971605
iGEM10_091 in KA2930iGEM10_091189831701605
iGEM10_092 in KA3132iGEM10_092198932611605
iGEM10_090 in CK3334iGEM10_09092821281473

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:

Layout:

' Lane # Part Name Part size Eco/xho Frags eco/Xho Frags
iGEM10_076 in AK1iGEM10_076187830781680
iGEM10_077 in CA2iGEM10_077187931511473
iGEM10_078 in CA3iGEM10_078152227941473
iGEM10_079 in CA4iGEM10_079111723891473
iGEM10_055 in KA5iGEM10_055146427361605
iGEM10_068 in AK6iGEM10_06877119711680
iGEM10_065 in CK7iGEM10_065255437541473
iGEM10_056 in AC8iGEM10_0568211481679
iGEM10_057 in CA9iGEM10_05785821301473
iGEM10_060 in AC10iGEM10_06036614321679
iGEM10_061 in CA11iGEM10_061106423361473
iGEM10_063 in CA12iGEM10_063105723291473
iGEM10_057 in CK13iGEM10_05785820581473
iGEM10_061 in CK14iGEM10_061106422641473
iGEM10_063 in CK15iGEM10_063105722571473
iGEM10_071 in KC16iGEM10_071190929751605
iGEM10_072 in KC17iGEM10_07269717631605
iGEM10_073 in CK18iGEM10_07399621961473
iGEM10_074 in CK19iGEM10_07449016901473
iGEM10_075 in CK20iGEM10_07549016901473
iGEM10_055 in KA21iGEM10_055146427361605
iGEM10_054 in CA22iGEM10_054133526071473
iGEM10_067 in CA23iGEM10_06715714291473
iGEM10_066 in CA24iGEM10_0668013521473

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

RE mapping of Stage 1 Catchups from Guildenstern Columns 5+7:
Gel:
Layout:

' ' ' Part Name Part size Eco/xho Frags eco/Xho Frags
iGEM10_062 in AC1,23,4iGEM10_06227913451679
iGEM10_079 in CA56iGEM10_079111723891473
iGEM10_065 in CK78iGEM10_065255437541473

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:
    • Part B:
  • Gel # 2 (Clones A+B from Rosencrantz):
    • Part A:
    • Part B:
  • Gel # 3 (Guildenstern Columns 1+3):

Layout:

Clones A and B are Gel #1 ' ' ' ' ' '
Clones C and D are on Gel #2
Lanes Clone A,CLanes Clone B,DPart NamePart sizeEco/xho Fragseco/Xho Frags
iGEM10_076 in AK12iGEM10_076187830781680
iGEM10_077 in CA34iGEM10_077187931511473
iGEM10_078 in CA56iGEM10_078152227941473
iGEM10_079 in CA78iGEM10_079111723891473
iGEM10_055 in KA910iGEM10_055146427361605
iGEM10_068 in AK1112iGEM10_06877119711680
iGEM10_064 in KC1314iGEM10_06499620621605
iGEM10_065 in CK1516iGEM10_065255437541473
iGEM10_056 in AC1718iGEM10_0568211481679
iGEM10_057 in CA1920iGEM10_05785821301473
iGEM10_060 in AC2122iGEM10_06036614321679
iGEM10_062 in AC2324iGEM10_06227913451679
iGEM10_066 in CA2526iGEM10_0668013521473
iGEM10_061 in CA2728iGEM10_061106423361473
iGEM10_063 in CA2930iGEM10_063105723291473
iGEM10_054 in CK3132iGEM10_054133525351473
iGEM10_057 in CK3334iGEM10_05785820581473
iGEM10_061 in CK3536iGEM10_061106422641473
iGEM10_063 in CK3738iGEM10_063105722571473
iGEM10_071 in KC3940iGEM10_071190929751605
iGEM10_072 in KC4142iGEM10_07269717631605
iGEM10_073 in CK4344iGEM10_07399621961473
iGEM10_074 in CK4546iGEM10_07449016901473
iGEM10_075 in CK4748iGEM10_07549016901473
Small Gel
iGEM10_055 in KA12iGEM10_055146427361605
iGEM10_054 in CA1,23,4iGEM10_054133526071473
iGEM10_067 in CA5,67,8iGEM10_06715714291473
iGEM10_066 in CA9,1011,12iGEM10_0668013521473

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

RE map of KA transfers:

Layout:

' Lane Lane part Part size Eco/xho Frags eco/Xho Frags
sbb19 in KA12sbb1959718691605
sbb18 in KA34sbb1839916711605
sbb10 in KA56sbb10102622981605
sbb42 in KA78sbb429113631605
sbb04 in KA910sbb04178830601605
sbb06 in KA1112sbb0624115131605
sbb04 in KA1314sbb04178830601605
Bjh2245 in KA1516Bjh22459713691605
Bjh1906 in KA1718Bjh19064713191605
sbb19 in KA1920sbb1959718691605
sbb12 in KA2122sbb1223415061605
sbb09 in KA2324sbb093513071605
sbb10 in KA2526sbb10102622981605
sbb42 in KA2728sbb429113631605
Bjh2294 in KA2930Bjh2294250737791605
sbb07 in KA3132sbb07143127031605

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

Checking some of the parts from Oliver twist:

Layout:

Lane # ' Plate Location part Part size Eco/xho Frags eco/Xho Frags
1sbb15 in ACFutureA1sbb159011561679
3sbb43 in AKFutureA4sbb4310013001680
5Bjh1906 in AKFutureB4Bjh19064712471680
7sbb13 in ACFutureB7sbb13181828841679
9Bjh2294 in AKFutureF10Bjh2294250737071680
11Bjh1858 in CAFutureA12Bjh18583313051473
2sbb15 in ACTiny TimA1sbb159011561679
4sbb43 in AKTube Cnasbb4310013001680
6Bjh1906 in AKCatA1Bjh19064712471680
8sbb13 in ACTiny TimC7sbb13181828841679
10Bjh2294 in AKCatA5Bjh2294250737071680
12Bjh1858 in CAScroogeA12Bjh18583313051473

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

Rechecked pMLL7 and pMLL4 for methylated parts:

Layout:

Lane # Part in vector opposite vector Part Vector Part Location in Tiny Tim Enzymes Expected bands Expected bands Wrong Bands Wrong Bands
1Bjh2294 in AKpMLL6-KABjh2294pMLL7-AKBjh2294Get from Chesire cat A 5EcoRI/Bgl1455483034791905
2Bjh1906 in CApMLL4-ACBjh1906pMLL9-CABjh1906A6EcoRI/Bgl1177310191962830
3sbb06 in AKpMLL6-KAsbb06pMLL7-AKsbb06G10EcoRI/Bgl1228883019051213
4sbb44 in CApMLL4-ACsbb44pMLL9-CAsbb44C6EcoRI/Bgl1179517732738830

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:

Gel Part B:

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 AK122324iGEM10_076187818453330781680
iGEM10_055 in KA342526iGEM10_055146414313327361605
iGEM10_068 in AK562728iGEM10_0687716819019711680
iGEM10_065 in CK782930iGEM10_065255447250737541473
iGEM10_073 in CK9103132iGEM10_07399639959721961473
iGEM10_054 in CK11123334iGEM10_054133512449125351473
iGEM10_057 in CK13143536iGEM10_0578588233520581473
iGEM10_061 in CK15163738iGEM10_061106482324122641473
iGEM10_063 in CK17183940iGEM10_063105782323422571473
iGEM10_074 in CK19204142iGEM10_0744909139916901473
iGEM10_055 in KA21224344iGEM10_055146414313327361605

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

Col PCR Stage 1 Round 3:
Part A:

Part B:

Part C:

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

Checking Vector Backbones Gel:

Lane # Part in vector Vector + Ebid Location in Romeo Enzymes Expected bands Expected bands Wrong Bands Wrong Bands
1sbb14 in AC4 + 17E8EcoRI/Bgl1376083028171773
2ig114 in CK5 + 8D5EcoRI/ScaI322169421461769
3sbb06 in KA6 + 5F1EcoRI/Bgl1190512132288830
4sbb17 in AK7 + 14E4EcoRI/Bgl1244683019051371
5sbb07 in KC8 + 4F6EcoRI/ScaI233317693408694
6sbb18 in CA9 + 13G9EcoRI/Bgl1177313712314830

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

Did colony PCR of Stage 1 Round 2assembly parts.

  • Gel #1
    • Part A:
    • Part B:
  • Gel #2
    • Part A:
    • Part B:



Layout for both gels:

' Lane ' '
Clone 1,3Clone 2,4Expected Band
iGEM10_076 in AK122078
iGEM10_055 in KA341664
iGEM10_068 in AK56971
iGEM10_064 in KC781196
iGEM10_065 in CK9102754
iGEM10_056 in AC1112282
iGEM10_057 in CA13141058
iGEM10_060 in AC1516566
iGEM10_062 in AC1718479
iGEM10_066 in CA1920280
iGEM10_061 in CA21221264
iGEM10_063 in CA23241257
iGEM10_054 in CK25261535
iGEM10_057 in CK27281058
iGEM10_061 in CK29301264
iGEM10_063 in CK31321257
iGEM10_071 in KC33342109
iGEM10_072 in KC3536897
iGEM10_073 in CK37381196
iGEM10_074 in CK3940690
iGEM10_075 in CK4142690
iGEM10_055 in KA43441664
iGEM10_054 in CA45461535
iGEM10_067 in CA4748357

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

Col PCR of oparts 77-79:

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.

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

Restriction map of Methylated Parts for stage 0 reruns:

Layout:

Re-runs ' ' ' ' ' '
sbb43 in AK12sbb4310013001680
ig114 in CA34ig114124425161473
Bjh1858 in CK56Bjh18583312331473
sbb07 in KA78sbb07143127031605
sbb20 in AK910sbb2059717971680

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

Restriction maps of Methylated Parts for Stage 0:

  • Lefty methylations:
    • Layout:
' Part A Lane # Part B Lane # Part Name Part size Eco/xho Frags eco/Xho Frags
sbb15 in AC12sbb159011561679
sbb14 in AC34sbb14184529111679
Bjh1882 in AC56Bjh188268117471679
sbb19 in KA78sbb1959718691605
sbb18 in KA910sbb1839916711605
sbb10 in KA1112sbb10102622981605
sbb42 in KA1314sbb429113631605
sbb04 in KA1516sbb04178830601605
sbb42 in CK1718sbb429112911473
sbb37 in CK1920sbb374112411473
Bjh1858 in CK2122Bjh18583312331473
ig114 in CK2324ig114124424441473
Bca1091 in CK2526Bca10916012601473
sbb44 in CK2728sbb4482320231473
sbb43 in AK2930sbb4310013001680
Bjh1906 in AK3132Bjh19064712471680
sbb14 in KC3334sbb14184529111605
sbb13 in KC3536sbb13181828841605
sbb07 in KC3738sbb07143124971605
Bjh1906 in CA3940Bjh19064713191473
sbb18 in CA4142sbb1839916711473
sbb44 in CA4344sbb4482320951473
sbb42 in CA4546sbb429113631473
ig114 in CA4748ig114124425161473
  • Righty Methylations:
    • Layout:
' Part A Lane # Part B Lane # Part Name Part size Eco/xho Frags eco/Xho Frags
sbb43 in AC12sbb4310011661679
sbb13 in AC34sbb13181828841679
sbb20 in AC56sbb2059716631679
sbb06 in KA78sbb0624115131605
sbb04 in KA910sbb04178830601605
Bjh2245 in KA1112Bjh22459713691605
sbb16 in CK1314sbb169012901473
Bjh1858 in CK1516Bjh18583312331473
Bjh1906 in KA1718Bjh19064713191605
sbb19 in KA1920sbb1959718691605
sbb12 in KA2122sbb1223415061605
sbb09 in KA2324sbb093513071605
sbb10 in KA2526sbb10102622981605
sbb42 in KA2728sbb429113631605
Bjh2294 in KA2930Bjh2294250737791605
sbb07 in KA3132sbb07143127031605
sbb18 in AK3334sbb1839915991680
sbb17 in AK3536sbb1739915991680
sbb12 in AK3738sbb1223414341680
sbb09 in AK3940sbb093512351680
sbb42 in AK4142sbb429112911680
Bjh2294 in AK4344Bjh2294250737071680
sbb06 in AK4546sbb0624114411680
sbb20 in AK4748sbb2059717971680
  • Extra Righties:
    • Layout:
' Part A Lane # Part B Lane # Part Name Part size Eco/xho Frags eco/Xho Frags
sbb08 in KC12sbb083511011605
sbb11 in KC34sbb1123212981605
sbb05 in KC56sbb0531913851605
Bjh1858 in CA78Bjh18583313051473

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:
Image:Re_map_of_igem_76.jpg

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

Colony PCR of manually assembled parts iGEM10_076-79:

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

Restriction map of catchup methylated parts:

Layout:

Part Strain Clone number Part number ' Lane Number Part size eco/xho frags eco/xho frags
8 with 1R4113513071473
9 with 3R4323310991605
8 with 1R3133513071473
9 with 3R3343310991605
8 with 1R2153513071473
9 with 3R2363310991605
8 with 1R1173513071473
9 with 3R1383310991605
------
9 with 23L42399111571605
5 with 20L4201082320231473
9 with 23L323119111571605
5 with 20L3201282320231473
9 with 23L223139111571605
5 with 20L2201482320231473
9 with 23L123159111571605
5 with 20L1201682320231473
Ladder--
9 with 11L411174611121605
8 with 21RB1211823215041473
9 with 11L311194611121605
8 with 21RB2212023215041473
9 with 11L211214611121605
8 with 21RB3212223215041473
9 with 11L111234611121605
8 with 21RB4212423215041473
------
9 with 20L4202582318891605
8 with 21RA1212623215041473
9 with 20L3202782318891605
8 with 21RA2212823215041473
9 with 20L2202982318891605
8 with 21RA3213023215041473
9 with 20L1203182318891605
8 with 21RA4213223215041473

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:

Layout of parts from plate A:

Parts from plate A ' ' ' ' '
PlasmidPart numberPart SizeEco/Xho FragsEco/Xho FragsLane #
5 with 2020823202314731
5 with 252541124114732
9 with 111146111216053
9 with 2020823188916054
9 with 232391115716055
7 with 1010100130016806
5 with 151590129014737
5 with 3333123314738
8 with 1135130714739
8 with 21212321504147310
4 with 10101001166167911
4 with 181818182884167912
4 with 995971663167913
9 with 33331099160514
Ladder
6 with 1111461318160515
7 with 13133991599168016
6 with 12125971869160517
7 with 14143991599168018
6 with 19192281500160519
7 with 19192281428168020
6 with 22351307160521
7 with 22351235168022
6 with 222210262298160523
7 with 2323911291168024
6 with 2323911363160525
7 with 242424003600168026
6 with 242424003672160527
7 with 552411441168028
6 with 4414312703160529
7 with 995971797168030

Col PCR map of basic parts:
Image: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 Bca1091Bca109160126014731
6 with Bjh2245Bjh224597136916052
6 with 1212597186916053
5 with 252541124114734
9 with 111146111216055
9 with 232391115716056

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

Restriction map of methylated parts Plate B:
Part 1:

Part 2:

Layout:

' RE mapping of ALL methylated parts, Plate B RE mapping plate layout: ' ' '
Ladder
4 with 161690115616791
5 with 2020823202314732
4 with 17171845291116793
5 with 232391129114734
4 with Bjh1882Bjh1882681174716795
5 with 252541124114736
9 with 111146111216057
5 with 3333123314738
9 with 1313399146516059
5 with 8812442444147310
9 with 20208231889160511
5 with Bca1091Bca1091601260147312
9 with 2323911157160513
8 with 171718453117147314
9 with 8812442310160515
8 with 181818183090147316
Ladder
6 with 12125971869160517
7 with 13133991599168018
6 with 13133991671160519
7 with 14143991599168020
6 with 222210262298160521
7 with 19192281428168022
6 with 2323911363160523
7 with 22351235168024
6 with 7717883060160525
7 with 2323911291168026
7 with 10101001300168027
7 with 242424003600168028
7 with 1111461246168029
7 with 552411441168030
8 with 4414312703147331
7 with 995971797168032
Ladder
6 with 1111461318160533
5 with 1515901290147334
6 with 12125971869160535
5 with 33331233147336
6 with 19192281500160537
8 with 11351307147338
6 with 22351307160539
8 with 21212321504147340
6 with 222210262298160541
8 with 663191591147342
6 with 2323911363160543
6 with 552411513160544
6 with 242424003672160545
6 with 7717883060160546
6 with 4414312703160547
6 with Bjh224515901362160548
Ladder
Gel #2
Ladder
4 with 1010100116616791
4 with 18333109916792
4 with 9181818288416793
9 with 3135110116054
6 with 24242400367216055
7 with 232391129116806
7 with 24242400360016807
9 with 111146111216058
9 with 232391115716059
5 with Bca1091Bca1091601260147310
4 with Bjh1882Bjh18826811747167911
6 with Bjh2245Bjh2245971369160512
6 with 12125971869160513
5 with 2525411241147314


Gel #2:

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

Restriction map of unmethylated parts:

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

Restriction Map of methylated parts plate rows A and E:

Layout:

' Part number Part Size Eco/Xho Frags Eco/Xho Frags Lane #
4 with 161690106617691
9 with 1313399106620042
5 with 2020823120022963
5 with 881244120027174
6 with 1212597127222025
6 with 771788127233936
7 with 1313399120020797
7 with 232391120017718
6 with 111146127216519
6 with 222210261272263110
5 with 1515901200156311
8 with 663191272179212
4 with 10101001066177913

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

Restriction Map Rerun:

Layout:

Restriction Mapping ' Lane #s A B Part size eco/xho frags eco/xho frags
9 with iGEM10_023R12187929451605
9 with iGEM10_024R34152225881605
9 with iGEM10_028L5615712231605
9 with iGEM10_025R7832313891605
9 with iGEM10_027L91085819241605
8 with iGEM10_029L1112190931811473
9 with iGEM10_034R21314106421301605
5 with iGEM10_035R151699621961473
7 with iGEM10_040R171877119711680
9 with iGEM10_008R1920105721231605
6 with iGEM10_033L2122146427361605

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

Colony PCR results from Group 2

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:

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:

Restriction Mapping of everything we've mini-prepped (groups 1 and 2)

Right Half

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:

' ' ' ' ' Lane # '
part size +200Eco/Bam fragsEco/Bam fragsAB
7 with iGEM10_022R20784755475512
9 with iGEM10_023R20794624462434
9 with iGEM10_024R17224267426756
9 with iGEM10_028L357157274578
4 with iGEM10_036R28228272827910
4 with iGEM10_037R566311131111112
4 with iGEM10_009R479302430241314
9 with iGEM10_025R523306830681516
9 with iGEM10_027L105885827451718
8 with iGEM10_029L2109190926711920
9 with iGEM10_026L1535133527452122
9 with iGEM10_027R1058360336032324

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_026L9 with 87 with 23
5 with iGEM10_027L9 with 207 with 2
5 with iGEM10_032L9 with 237 with 13
5 with iGEM10_034L9 with 207 with 5
5 with iGEM10_008L9 with 207 with 19
8 with iGEM10_035L6 with 134 with 9
5 with iGEM10_034R9 with 207 with 5
5 with iGEM10_008R9 with 207 with 19
8 with iGEM10_035R6 with 134 with 9
  • Assembled these parts that we missed/plated on the wrong antibiotics manually:
Name Strain Lefty Input Righty Input
5 with iGEM10_035L9 with 137 with 9
6 with iGEM10_033L8 with 49 with 3
9 with iGEM10_034L5 with 206 with 5
9 with iGEM10_008L5 with 206 with 19
5 with iGEM10_035L9 with 137 with 9
5 with iGEM10_038R9 with 117 with 24
7 with iGEM10_040R4 with Bjh18825 with 15
6 with iGEM10_033R8 with 49 with 3
9 with iGEM10_034R5 with 206 with 5
9 with iGEM10_008R5 with 206 with 19
5 with iGEM10_035R9 with 137 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


and Plate B

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)

and Part 2

Here are columns 7-10:

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_026L9 with 87 with 23
5 with iGEM10_027L9 with 207 with 2
5 with iGEM10_032L9 with 237 with 13
5 with iGEM10_034L9 with 207 with 5
5 with iGEM10_008L9 with 207 with 19
8 with iGEM10_035L6 with 134 with 9
5 with iGEM10_034R9 with 207 with 5
5 with iGEM10_008R9 with 207 with 19
8 with iGEM10_035R6 with 134 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:

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.

Looks ok. Added these parts to methylated parts plates

Lane # ID Name Expected fragments '
14 with 10 #1pMLL4-AC+B10sbb43 8501990
24 with 10 #2pMLL4-AC+B10sbb43 8501990
34 with 18 #1pMLL4-AC+B10sbb13 8502160
44 with 18 #2pMLL4-AC+B10sbb13 8502160
54 with 9 #1pMLL4-AC+B10Sbb208502450
64 with 9 #2pMLL4-AC+B10Sbb208502450
79 with 3 #1pMLL9-CA+Bjh18533001500
89 with 3 #2pMLL9-CA+Bjh18533001500

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
A4 with 165 with 206 with 127 with 108 with 179 with 114 with 106 with 116 with 57 with 138 with 19 with 3
B4 with 175 with 236 with 137 with 118 with 189 with 134 with 186 with 126 with 77 with 148 with 21
C4 with Bjh18825 with 256 with 228 with 49 with 204 with 96 with 196 with Bjh22457 with 198 with 6
D5 with 36 with 239 with 236 with 27 with 2
E5 with 86 with 79 with 86 with 227 with 23
F6 with Bca10916 with 237 with 24
G5 with 156 with 247 with 5
H5 with 36 with 47 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
A6+26+116+227+97+245+238+174+109+39+207+24
B6+26+116+227+105+35+238+174+109+39+209+11
C6+46+126+237+115+38+18+184+169+89+239+23
D6+46+126+237+135+88+18+184+169+89+23pMLL5+Bca1091 (L)
E6+56+136+247+145+88+48+214+179+11pMLL4+Bjh1882 (L)
F6+56+136+247+195+158+48+214+179+11pMLL6+Bjh2245
G6+76+197+27+235+208+64+94+189+136 +246+12
H6+76+197+57+245+208+64+94+189+137 + 235+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
A4 with 165 with 206 with 127 with 108 with 179 with 114 with 106 with 116 with 57 with 138 with 19 with 3
B4 with 175 with 236 with 137 with 118 with 189 with 134 with 186 with 126 with 77 with 148 with 21
C4 with Bjh18825 with 256 with 228 with 49 with 204 with 96 with 196 with Bjh22457 with 198 with 6
D5 with 36 with 239 with 236 with 27 with 2
E5 with 86 with 79 with 86 with 227 with 23
F6 with Bca10916 with 237 with 24
G5 with 156 with 247 with 5
H5 with 36 with 47 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.


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
A6+26+116+227+97+245+238+174+109+39+207+24Lysis Device
B6+26+116+227+105+35+238+174+109+39+209+115+25
C6+46+126+237+115+38+18+184+169+89+239+23
D6+46+126+237+135+88+18+184+169+89+23pMLL5+Bca1091 (L)
E6+56+136+247+145+88+48+214+179+11pMLL4+Bjh1882 (L)
F6+56+136+247+195+158+48+214+179+11pMLL6+Bjh2245
G6+76+197+27+235+208+64+94+189+136 +246+12
H6+76+197+57+245+208+64+94+189+137 + 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 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:

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:

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 ' ' ' '
18821091 #11091 #2
AAA
BBB
CCC
DDD
  • 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 ' ' ' '
18821091 #11091 #2
AAA
BBB
CCC
DDD

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 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.

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