IGEM:MIT/2006/Notebook/2006-6-28

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)

Update on cultures

Created cultures of BSMT and SAMT with 40 uL .5 M SA. They again smelled. Our intial idea was to add MS to some of the cultures in different amounts to qualitatively estimate how much MS the cells treated with the plasmids were producing. However, we had a very difficult time diluting the concentrated samples of MS and MB down. We tried with DMSO, and the solution dissolved the sterilization filter. We tried with water, but the solution kept phase separating. We tried first dissolving in ethanol and then in water, but again, the solution phase separated.

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...

1. 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!

Sequenced Redone SAMT Primer

1. Protocol on Index page

BSMT PCR Product Eco/Pst Digest

1. 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
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
   • BSMT pcr product at 50 ng/μL -----add 16 μL
   • control part PSBI82E0040 at 200 ng/μL ----add 4 μL
4. 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)
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. BSMT pcr cleanup product cut reaction (in order added, following the Knight Lab restriction digest protocol):
   • 26.5 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
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 !!