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)

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!

SAMT sequencing

1. our new internal primer has arrived, lets try to sequence again

Restriction Digest--TAKE 2

things that we did the first time (that we changed): we used a Kan backbone (this time we used the CHL one), we used a different part from Barry (control part 3k3.I7101 at 44 ng/μL -- we added 20 μL), we added 9μL of BSMT b/c it's concentration was 90ng/μL, and we added less backbone (I think 28μL?)

1. We are cutting the backbone and the BSMT pcr product separately (using EcoRI and PstI)
2. Total reaction volume is 50μL
3. Our control cut reaction is with the part PSBI82E0040 from Barry
4. the total volume desired is about 50 μL and amount of dna desired is about 800 nanograms (it was 400ng last time, but Samantha said 1mg so we doubled.)
   • pSBIAC3 backbone at 20 ng/μL -----add 40 μL backbone
   • BSMT pcr product at 50 ng/μL -----add 16 μL
   • control part PSBI82E0040 at 200 ng/μL ----add 4 μL
5. we are also going to use Dpn1 to destroy all dna that is not a pcr product (i.e. get rid of extra template)
6. BACKBONE cut reaction (in order added, following the Knight Lab restriction digest protocol):
   • 3 μL water
   • 5 μL NEB buffer 2
   • .5 μL BSA
   • 40 μL backbone plasmid
   • .5 μL Pst1
   • .5 μL EcoR1
   • .5 μL Dpn 1
7. BSMT pcr cleanup product cut reaction (in order added, following the Knight Lab restriction digest protocol):
   • 34 μL water
   • 5 μL NEB buffer 2
   • .5 μL BSA
   • 16 μL BSMT pcr cleanup product (concentration = 50 ng/μL)
   • .5 μL Pst1
   • .5 μL EcoR1
   • .5 μL Dpn 1
8. 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)
   • .5 μL Pst1
   • .5 μL EcoR1
   • .5 μL Dpn 1
9. Incubate both tubes in 37c room for 4-6 hours, then 20 mins at 80deg C to heat inactivate the enzyme, then 4deg forever.