Berk2010-Tahoura: Difference between revisions
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==[[User:Tahoura Samad|Tahoura Samad]] 17:20, 9 July 2010 (EDT)== | ==[[User:Tahoura Samad|Tahoura Samad]] 17:20, 9 July 2010 (EDT)== | ||
[[Image:[[IMG_5245.JPG | [[Image:[[IMG_5245.JPG]] | ||
=[[User:Tahoura Samad|Tahoura Samad]] 15:18, 8 July 2010 (EDT)= | =[[User:Tahoura Samad|Tahoura Samad]] 15:18, 8 July 2010 (EDT)= | ||
==PDD Subgroup Work== | ==PDD Subgroup Work== |
Revision as of 14:20, 9 July 2010
Tahoura Samad 17:20, 9 July 2010 (EDT)
[[Image:IMG_5245.JPG
Tahoura Samad 15:18, 8 July 2010 (EDT)
PDD Subgroup Work
- direct Jin to confirm which of the combinations is actually working.
- remind Tim to email Jin about scope.
Life Act
- Even after 24 hours, the bacteria from the colony PCR still did not grow up well. Only saw growth for a couple. Miniprepped colonies 6, 9, 19, 20. Restriction mapped Bam, Eco, excpected band length of 167 and 2700
Other
- picked MC1061 pir1 lefty and righty and am growing up in LB.
Tahoura Samad 16:10, 7 July 2010 (EDT)
PDD Subgroup Work
- nothing
LifeAct
- redid colony PCR with 24 new colonies. Results looked good, with many bands at expected length of 357.
- will miniprep in the afternoon.
Other
- streaked out MC1061 pir lefty and righty since we're almost out. Will make competent cells on Friday.
To Do
- Pick and grow up Pir Lefty and Righty for competent cell prep on Friday.
- On Friday, do transformations of all 3s with all five PDDs.
- On Friday, make competent cells.
- On Saturday, take out of incubator, pick and grow up.
- On Sunday, feed to choanos.
- On Sunday, pick and grow up 3 and 5 for Monday.
- ask Tim what to do with IBB.
- get solutions of MMM and MM0 from Jin.
- ask Jin #grow up in shaker for 2 more hours. (Ask Jin what how much of each solution to grow them up in.)
- split choanos 1:5 on Sunday, so we'll have enough for Monday. Check with conor how to get 17 mL of choanos for Monday.
Tahoura Samad 18:16, 6 July 2010 (EDT)
PDD Subgroup Work/Discussion With Jin
- talked to Jin. He was not able to look at the combinations from Friday. :(
- Jin says to let all the different combinations rest, and focus on the 1934 payload.
- Jin also says to start the minimal media testing to see if pb6 works.
Minimal Media Testing
- Day 1
- Streak out 3, 5
- Pick and grow up in TB with 30 mM MgS04 (30 μL per mL) overnight.(16 hrs) Do two colonies of each 3 and 5.
- Day 2
- Dilute 100 fold by adding 30 μL of overnight culture to 3 mL of TB. (4 tubes, 2 of 3 and two of 5)
- Grow for 2-3 hours more. (2 hours)
- make 2. 1 mL aliquots of 3 and 5. (Four of each) (8 tubes total)
- Wash 3 times.
- Wash two of each with minimal media with magnesium (MMM) and Minimal media without magnesium. (MM0) Get these solutions from Jin.
- When you wash these guys, spin the cells down, carefully pipette off the supernatant without touching the pellet, resuspend with solution etc .3 times.
- grow up in shaker for 2 more hours. (Ask Jin what how much of each solution to grow them up in.)
- filter 17 mL of choanos
- incubate 8 mL of choanos with 10 μL per mL (10 mM ) NH4Cl for 10 minutes.
- feed 10 μL of bacteria to choanos. Look at after 5 hours.
Lifeact
- ran colony PCR for 32 colonies.
- minipreped 30 and 32, but they turned out to be contransformed. Of 32 colonies, 7 were contransformed.
NLS
- sequencing results looked okay.
- iGEM_028, CK lefty looks perfect except for a point mutation in the EcoR1 site. Looking at the abi, i think it might be a miscall.
- iGEM_029, CK Righty looks perfect except for a one base deletion after the Pts1 site. Looking at the Abi file, exactly at this point, the sample gets mixed reads. ?
- iGEM_030, CA Righty looks perfect except for the point mutation in the Ecor1 site. Looking at the abi, i think it might be a miscall.
To Do
- On Friday, do transformations of all 3s with all five PDDs.
- On Saturday, take out of incubator, pick and grow up.
- On Sunday, feed to choanos.
- On Sunday, pick and grow up 3 and 5 for Monday.
Tahoura Samad 13:22, 5 July 2010 (EDT)
- picked and grew up 1, 2, 6, 7, to make competent cells.
Tahoura Samad 13:22, 2 July 2010 (EDT)
- most of my transformations with the new competent cells were successful.
Competent cell | Number | PDD | Growth in initial competent cells | Growth in Small Scale Competent Cells | Result of Assay with Jin | Conclusion | |
pBca156-Bjh2343 | 1 | 2331 | 0 | tons of colonies | NOT DONE | transform new, assay | |
pBca156-Bjh2343 | 1 | 2333 | 0 | tons of colonies | NOT DONE | transform new, assay | |
pBca156-Bjh2343 | 1 | 2348 | 1 colony (twice) | tons of colonies | NOT DONE | transform new, assay | |
pBca156-Bjh2343 | 1 | 2349 | 0 | 50-100 colonies | NOT DONE | transform new, assay | |
pBca156-Bjh2343 | 1 | 2291 | 0 | 100-200 colonies | NOT DONE | transform new, assay | |
pBca156-Bjh2343 with BAC | 7 | 2331 | 0 | 15 colonies | NOT DONE | Today | |
pBca156-Bjh2343 with BAC | 7 | 2333 | 0 | 2 colonies | NOT DONE | Today | |
pBca156-Bjh2343 with BAC | 7 | 2348 | 0 | 4 colonies | NOT DONE | Today | |
pBca156-Bjh2343 with BAC | 7 | 2349 | 0 | 0 | DEAD | \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ | |
pBca156-Bjh2343 with BAC | 7 | 2291 | 0 | 0 | DEAD | \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ | |
pBca156-Bjh2344 | 2 | 2331 | 0 | tons of colonies | NOT DONE | make new, transform, assay | |
pBca156-Bjh2344 | 2 | 2333 | 0 | tons of colonies | NOT DONE | make new, transform, assay | |
pBca156-Bjh2344 | 2 | 2348 | 12 colonies | TO DO | V (UNSTABLE) | make new, transform new and old, assay | |
pBca156-Bjh2344 | 2 | 2349 | 6 colonies | TO DO | V V | make new, transform new and old, assay | |
pBca156-Bjh2344 | 2 | 2291 | 7 colonies | TO DO | V V | make new, transform new and old, assay | |
pBca156-Bjh2344 with BAC | 6 | 2331 | tons of colonies | X | X | \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ | |
pBca156-Bjh2344 with BAC | 6 | 2333 | tons of colonies | X | X | \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ | |
pBca156-Bjh2344 with BAC | 6 | 2348 | 1 colony (twice) | 100-200 colonies | NOT DONE | Today | |
pBca156-Bjh2344 with BAC | 6 | 2349 | 1 colony(twice) | 35-50 colonies | X | \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ | |
pBca156-Bjh2344 with BAC | 6 | 2291 | 35-50 colonies | 100-200 colonies | V V V | transform new, assay | |
pBca156-Bjh1934 | 3 | 2331 | 35-50 colonies | X | X | \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ | |
pBca156-Bjh1934 | 3 | 2333 | <100 colonies | X | X | \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ | |
pBca156-Bjh1934 | 3 | 2348 | tons of colonies | X | V V | transform old, assay | |
pBca156-Bjh1934 | 3 | 2349 | tons of colonies | X | V V | transform old, assay | |
pBca156-Bjh1934 | 3 | 2291 | tons of colonies | X | V | transform old,assay | |
pBca156-Bjh1934 with BAC | 5 | 2331 | tons of colonies | X | X | \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ | |
pBca156-Bjh1934 with BAC | 5 | 2333 | tons of colonies | X | X | \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ | |
pBca156-Bjh1934 with BAC | 5 | 2348 | tons of colones | X | X | \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ | |
pBca156-Bjh1934 with BAC | 5 | 2349 | 1o colonies | X | X | \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ | |
pBca156-Bjh1934 with BAC | 5 | 2291 | tons of colonies | X | V V V | transform old, assay |
- competent cell checks for new 1 and new 6 looked good.
- miniprepped IBB as CA lefty, CK lefty and CK righty, and sent out for sequencing with ca998.
Tahoura Samad 13:38, 1 July 2010 (EDT)
- controls for JTK030 looked good with no growth on Kan, Amp, Cam, good growth on LB, and a little growth on Spec, which is the normal result because of mutations in the bacteria. Those are labeled with double black stripes, in a box in the back of the -80 in the 2nd row from the top.
- picked and grew up the results of PCA for the new nuclear localization tag. They had plenty of colonies, with only one background green colony. Will miniprep them tomorrow.
- did small scale competent cell prep of 1 and 6.
- did the following transformations (1old:2348), (5 old:2291) (6 old:2291) (6 new:2291),(3 old :2348) (3 old: 2349) (3 old:2291) (2 old:2348) (2 old: 2349)(2 old:2291) (2 new:2348) (2 new:2349) (2 new: 2291), (1 new:2331) (1 new:2333) (1 new:2348) (1 new:2349) (1 new: 2291) (6 new 2348) (6 new:2349), (IBB into lefty).
- redid assembly of stage 0 of life act but with 2.4 μL of each DNA and 1.2 μL of 5x NEB2.