IGEM:MIT/2006/Notebook/2006-6-30

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.

ATF1 PCR Product Eco/Pst Digest

1. We are cutting the (uncut, non-pcr product) backbone pSB1A3-1 and the ATF1 pcr product separately (using EcoRI and PstI) as well as Barry's (non-pcr product) control part pSBI82E0040
2. Total reaction volumes are 50μL
3. the amount of dna desired is about 800 nanograms (it was 400ng last time, but Samantha said 1microg so we doubled.)
   • pSBIA3-1 backbone at 100 ng/μL -----add 8 μL backbone
   • ATF1 pcr product at 30.8 ng/μL -----add 26 μL
   • control part PSBI82E0040 at 200 ng/μL ----add 4 μL
4. we are also going to use Dpn1 only in ATF1 cut reaction to destroy dna that is not a pcr product (i.e. get rid of extra template)
5. BACKBONE cut reaction (in order added, following the Knight Lab restriction digest protocol):
   • 35 μL water
   • 5 μL NEB buffer 2
   • .5 μL BSA
   • 8 μL backbone plasmid
   • .75 μL Pst1
   • .75 μL EcoR1
6. ATF1 pcr cleanup product cut reaction (in order added, following the Knight Lab restriction digest protocol):
   • 16.5 μL water
   • 5 μL NEB buffer 2
   • .5 μL BSA
   • 26 μL BSMT pcr cleanup product (concentration = 50 ng/μL)
   • .75 μL Pst1
   • .75 μL EcoR1
   • .5 μL Dpn 1
7. CONTROL (Barry's biobrick) cut reaction (in order added, following the Knight Lab restriction digest protocol):
   • 39 μL water
   • 5 μL NEB buffer 2
   • .5 μL BSA
   • 4 μL part PSBI82E0040 (from Barry)
   • .75 μL Pst1
   • .75 μL EcoR1
8. Incubate both tubes 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 !!

SAMT PCR Product Eco/Pst Digest

1. We are cutting the (uncut, non-pcr product) backbone pSB1A3-1 and the SAMT pcr product separately (using EcoRI and PstI) as well as Barry's (non-pcr product) control part pSBI82E0040
2. Total reaction volumes are 50μL
3. the amount of dna desired is about 800 nanograms (it was 400ng last time, but Samantha said 1microg so we doubled.)
   • pSBIA3-1 backbone at 100 ng/μL -----add 8 μL backbone
   • SAMT pcr product at 75.5 ng/μL -----add 11 μL
   • control part PSBI82E0040 at 200 ng/μL ----add 4 μL
4. 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)
5. BACKBONE cut reaction (in order added, following the Knight Lab restriction digest protocol):
   • 35 μL water
   • 5 μL NEB buffer 2
   • .5 μL BSA
   • 8 μL backbone plasmid
   • .75 μL Pst1
   • .75 μL EcoR1
6. 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
7. CONTROL (Barry's biobrick) cut reaction (in order added, following the Knight Lab restriction digest protocol):
   • 39 μL water
   • 5 μL NEB buffer 2
   • .5 μL BSA
   • 4 μL part PSBI82E0040 (from Barry)
   • .75 μL Pst1
   • .75 μL EcoR1
8. Incubate both tubes 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 !!