Team:BYU Provo/Team Thermosensor/Week11

Week 11 (June 26 - Jul 2)

6/27/11

It looks like 33-1 worked, but I don’t know if lacZ and GFP were actually inserted

Doesn’t look like lacZ did, but GFP looks promising.

6/28/11

PCR purfication of 33-1 (lacZ PCR)

• Qiaquick PCR purification kit protocol -Julie

lacZ restricition digest (TRD 34-1)

• T33-1 PCR up/lacZ with 1 ul of HindIII and XbaI each

pPLAT-Bad restriciton digest (TRD 34-2)

• pIG 13 with 1 ul each of HindIII and XbaI

Ran a low melt. Cut out pPLAT-Bad and stuck in 1.5 centrifuge tube. Could not see lacZ band...->doesn’t matter, I used wrong lacZ reverse

We could run another low melt and we may want to PCR it again. Other half of low melt in fridge.

lacZ PCR (35-1)

• Phusion polymerase and 1 ul each of IG40 and IG41 primers with pIGGY template
• Making two in case there is another purification problem or something

35-2: PCR up of lacZ according to qiaquick instructions

6/30/11

Running gel on lacZ PCR products

• Both lanes look good. Now onto restriction digest.

RD lacZ (35-3)

• 1-2 ul each restricition enzyme (HindIII and XbaI)

35-4 Ran low melt and cut out lacZ band. Very faint, but there!

lacZ/pPlatbad ligation 36-1

• T4 ligase, 3 ul vector, 3 ul lacZ (added a little extra b/c faintness of band)

7/1/11

lacZ/pPlatbad transformation

Followed Grose protocol

Plated 100 ul on LB-amp and incubated overnight

Labeled T36-1 lacZ trans 1 July 2011 - Devin