IGEM:MIT/2006/Notebook/2006-6-28
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Ligation (ONLY IF DIGEST WORKS, but digest didn't work so IGNORE THIS for now)
We'll do 2 ligations: one with the cut CHL backbone and our cut BSMT DNA, and another one with the cut CHL backbone and Barry's cut biobrick part.
Note to Andre: the 3 tubes of cut DNA are in the freezer in the neon yellow rack
General ligation considerations: 10μL total volume, 50ng of each DNA (but extra insert can't hurt)
BSMT Ligation
- 3μL water
- 1μL T4 DNA ligase buffer (vortex well before adding, make sure it smells strongly like wet dog!!)
- .5μL T4 DNA ligase enzyme
- 3μL cut BSMT (70ng)
- 2.5μL cut backbone (50ng)
Control Ligation (Barry's biobrick part)
- 3μL water
- 1μL T4 DNA ligase buffer (vortex well before adding, make sure it smells strongly like wet dog!!)
- .5μL T4 DNA ligase enzyme
- 3μL cut Barry's BB part (90ng)
- 2.5μL cut backbone (50ng)
Transformation (IGNORE THIS FOR NOW TOO)
Think about doing something to see if the ligation worked before transforming...perhaps run a gel? Find out if there's anything else we can do...
- Thaw Tom's Top10 cells (don't use ones that have been thawed and refrozen)...then proceed with the transformation like we normally do (30 min incubate on ice, 50 sec heat shock, 1 hr incubation, then maybe something else?, then plating on the CHL/Amp plates). Don't forget to transform pUC19 : ) yay positive controls!
SAMT sequencing
- our new internal primer has arrived, lets try to sequence again
pure wintergreen testing
Restriction Digest--TAKE 3
- We are cutting the (uncut, non-pcr product) backbone pSB1A3-1 and the BSMT pcr product separately (using EcoRI and PstI) as well as Barry's (non-pcr product) control part pSBI82E0040
- Total reaction volumes are 50μL
- 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
- BSMT pcr product at 50 ng/μL -----add 16 μL
- control part PSBI82E0040 at 200 ng/μL ----add 4 μL
- we are also going to use Dpn1 only in BSMT cut reaction to destroy dna that is not a pcr product (i.e. get rid of extra template)
- 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
- BSMT pcr cleanup product cut reaction (in order added, following the Knight Lab restriction digest protocol):
- 33.5 μL water
- 5 μL NEB buffer 2
- .5 μL BSA
- 16 μL BSMT pcr cleanup product (concentration = 50 ng/μL)
- .75 μL Pst1
- .75 μL EcoR1
- .5 μL Dpn 1
- 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
- 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 to see if cutting worked!!!!