Team:BYU Provo/Team Thermosensor/Week10

Week 10 (June 19 - 25)

20 June 2011

It looks like we mixed something up. Lane 2 was marked as pLATBAD but appears to actually be the LacZ insert. Lane 3 was supposed to be the LacZ insert but appears to actually be the pLATBAD vector. We cut out the gel slices with the respective inserts or vector and performed ligations inserting LacZ and GFP between the HindIII/XbaI sites pLATBAD. (3 ul vector and insert for both)

Then the gel slices were placed in a 65oC heat block at 3:05pm to prepare for transformation.

Transformation performed and incubated at 37o overnight following Grose protocol.

Labeled: T30-1 (T30-1 lig/vector+lacZ) and T30-2 (T30-2 lig/vector+GFP)

6/21/11

No growth on T30-1. Only one potential colony on T30-2. Placed both in refrigerator in 888. -Addison

6/22/11

Retry of transformation (same protocol)

We’re going to run a gel on lacZ, GFP, and a few thermosensor PCRs to make sure we have good product

• Looks like good PCR product
• T22-4 may have gotten lost in clean up or somewhere. T19-1 was a good lacZ PCR product but I was unable to find it

6/23/11

Colonies grew on both GFP plate (30-2) and lacZ plates (30-2) (new plates from 6/22)

Boiled colonies in preparation for colony PCR

• lacZ: 30-1-(1-8)
• GFP: 30-2-(1-8)

6/24/11

lacZ colony PCR

• Taq polymerase protocol. 9 ul of IG40 and IG12 primers

GFP colony PCR

• Taq polymerase. 9 ul of IG26, IG12

PCR on lacZ (33-1)

• Phusion polymerase. 2 ul of each primer (IG39 and IG40) because they were more dilute

Next step -> run all on gel to check if have product