IGEM:MIT/2006/Notebook/2006-6-30: Difference between revisions
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==SAMT PCR Product Eco/Pst Digest== | ==SAMT PCR Product Eco/Pst Digest== | ||
#We are cutting the SAMT pcr product (using EcoRI and PstI) ''Began at 11AM'' | #We are cutting the SAMT pcr product (using EcoRI and PstI)--'''Began at 11AM''' | ||
#Total reaction volume is 50μL | #Total reaction volume is 50μL | ||
#the amount of dna desired is about 800 nanograms (it was 400ng last time, but Samantha said 1microg so we doubled.) | #the amount of dna desired is about 800 nanograms (it was 400ng last time, but Samantha said 1microg so we doubled.) | ||
Revision as of 08:04, 30 June 2006
Stationary Phase Promoter Stuff To Get Done
Two plates carrying the control to compare with the stationary phase promoter are in the 30 degree room. Remove them at the beginning of the day (really early if you don't want to come in on Saturday) and inoculate them in LB AMP liquid culture. Please remember to take them out. There is also a liquid culture labelled "control promoter" in the 37 degree room. I don't think it was growing yesterday, but you may want to check if it grew. If it grew, you still may want to make the LB AMP liquid culture from the fresh plates. If it didn't, I guess we have to make the liquid cultures from the 30 degree plates. If you guys feel ambitious and the liquid culture grew, you could start the fluorescent experiments (you can ask Barry to show you how it's done). Anyways, good luck, and have a great weekend!
Number of Transformants from 6/29 BSMT-BB Ligation/Transformation
| pUC19-Old Top 10 | pUC19-Fresh Top 10 | E0040BB (+Ctrl) | BSMTBB | |
|---|---|---|---|---|
| 20μL | 0 | 0 | 6 | 1 |
| 200μL | 2 | 20 | ~100 | 5 |
We can make a few conclusions from these data:
- The old Top 10 cells are probably not suitable for use (despite colonies appearing at the 200μ plating volume).
- Barry's E0040 positive control worked.
- Our ligation was "succcessful", as colonies did appear on our plates.
SAMT PCR Product Eco/Pst Digest
- We are cutting the SAMT pcr product (using EcoRI and PstI)--Began at 11AM
- Total reaction volume is 50μL
- the amount of dna desired is about 800 nanograms (it was 400ng last time, but Samantha said 1microg so we doubled.)
- SAMT pcr product at 75.5 ng/μL -----add 11 μL
- we are also going to use Dpn1 only in SAMT cut reaction to destroy dna that is not a pcr product (i.e. get rid of extra template)
- SAMT pcr cleanup product cut reaction:
- 31.5 μL water
- 5 μL NEB buffer 2
- .5 μL BSA
- 11 μL BSMT pcr cleanup product (concentration = 50 ng/μL)
- .75 μL Pst1
- .75 μL EcoR1
- .5 μL Dpn 1
- Incubate tube in 37c room for 1-6 hours, then 20 mins at 80deg C to heat inactivate the enzyme, then 4deg forever. ---- and run a gel before moving on to ligation !!
