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