Berk2010-Christoph: Difference between revisions
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==[[User:Christoph Neyer|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. | |||
*4 with 10 = pMLL4-AC+B10sbb43 | |||
**Plasmid with part = 2845bp | |||
**Fragment Digest Sizes = 850 and 1990 | |||
*4 with 18 = pMLL4-AC+B10sbb13 | |||
**Plasmid with part = 4563bp | |||
**Fragment Digest Sizes = 850 and 2160 | |||
*4 with 9 = pMLL4-AC+B10Sbb20 | |||
**Plasmid with part = 2845bp | |||
**Fragment Digest Sizes = 850 and 2450 | |||
*9 with 3 = pMLL9-CA+Bjh1853 | |||
**Plasmid with part = 2845bp | |||
**Fragment Digest Sizes = 300 and 1500 | |||
==[[User:Christoph Neyer|Christoph Neyer]] 21:36, 11 June 2010 (EDT)== | ==[[User:Christoph Neyer|Christoph Neyer]] 21:36, 11 June 2010 (EDT)== | ||
Plated Transformations from robot dilution. <br> | Plated Transformations from robot dilution. <br> | ||
==[[User:Christoph Neyer|Christoph Neyer]] 19:04, 11 June 2010 (EDT)== | ==[[User:Christoph Neyer|Christoph Neyer]] 19:04, 11 June 2010 (EDT)== | ||
Next step is to transform each part into a Righty or Lefty strain. <br> | Next step is to transform each part into a Righty or Lefty strain. <br> |
Revision as of 11:18, 14 June 2010
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.
- 4 with 10 = pMLL4-AC+B10sbb43
- Plasmid with part = 2845bp
- Fragment Digest Sizes = 850 and 1990
- 4 with 18 = pMLL4-AC+B10sbb13
- Plasmid with part = 4563bp
- Fragment Digest Sizes = 850 and 2160
- 4 with 9 = pMLL4-AC+B10Sbb20
- Plasmid with part = 2845bp
- Fragment Digest Sizes = 850 and 2450
- 9 with 3 = pMLL9-CA+Bjh1853
- Plasmid with part = 2845bp
- Fragment Digest Sizes = 300 and 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.
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 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 | ' | ' | ' | ' |
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 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.