IGEM:MIT/2006/Notebook/2006-6-29

PCR cleanup / Nanodrop / ran gel - TAKE 3

1. PCR cleanup of BSMT and Barry's part: followed standard protocol for PCR cleanup...not too exciting : ) Just remember to elute with 30μL of the last buffer.
2. Nanodrop: check the concentrations of the cleaned-up cut DNA, and of the cut backbone:
   • Plasmid (pSB1A3-1, cut with EcoRI and PstI): ? ng/μL
   • Barry's part (PSBI82E0040, cut with EcoRI and PstI): ? ng/μL
   • BSMT (cut with EcoRI, PstI, and DpnI): ? ng/μL
3. Gel: the columns, from left to right, are:
   • 2log ladder
   • cut BSMT (100ng) ... expected length = 1.1kB
   • cut Barry's biobrick part (100ng) ... expected length = 2kB backbone + .72kB fragment
   • cut pSB1A3-1 backbone (lots and lots b/c we have to gel extract) ... expected length = 2.1kB + another short fragment
   • uncut pSB1A3-1 backbone (2μL, aka 200ng)... expected to look crazy b/c it's a circle

Ligation (ONLY IF DIGEST WORKS, but digest didn't work so IGNORE THIS for now)

We'll do 2 ligations: one with the cut pSB1A3-1 backbone and our cut BSMT DNA, and another one with the cut pSB1A3-1 backbone and Barry's cut biobrick part.

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)