Team:Nevada/Week 6

June 25

Jasmine:

Cultured more EP in TB-Amp

Michelle and Joe:

Re-run PCR colony samples of RFP-TA colony #2 (XbaI and PstI) transformation from 6/22.

Cultured colonies #7, 8, 9 from PCR colony samples RFP-TA colony #2 (XbaI and PstI) with TB-AMP.

Transformation of ligation of SBP + LRP (XbaI, EcoRI, SpeI, and PstI) with EP from 6/22 on X-gal + AMP plate.

Jeremiah & Chris:

Transformation of SBP-TBP-VB12

Justin and Dafne:

At this point, the unsuccessful nature of our project lead to the restart from the point of ligating SBP-B12 insert into the expression plasmid

New Miniprep buffers were used

New ligase was used

June 26

Jasmine:

Miniprepped new EP cultures

Digested EP with SpeI

Michelle and Joe:

Check transformation of SBP + LRP (XbaI, EcoRI, SpeI, and PstI) with EP on X-gal + AMP plate.

June 27

Jasmine, Joe, and Michelle:

Run EP digest on agarose gel to check

Created primer stocks for new RFP (RFP*), new EP (EP*), and new controlled promoter (CP): RFP*-sense-SPEI-NsiI, RFP*-anti-PstI, EP*-sense-NsiI, EP*-anti-SpeI, CP-sense-EcoRI, CP-anti-SpeI

Amplified EP* using Phusion PCR

Blunted EP* with Blunting Enzyme

Jeremiah & Chris:

Colony PCR of transformation from 6/25 (gel 040) – good yield on all lanes

Cultured samples 1 & 8

Phusion PCR of SBP-TBP in expression vector

Justin and Dafne:

SBP “fusion” into SBP-B12 plasmid.

SBP-B12 was digested using Xba I and Pst I HF

PCR was used to add 15 bp to the end of both SBP and the open SBP-B12 plasmid

This process allowed for the annealing of SBP and SBP-B12 to occur without the normal ligation process

SBP-B12 insert was also ligated into the new expression plasmid using this technique

June 28

Jasmine, Joe, and Michelle:

Self-ligated blunted EP*

Transformed EP* into competent cells and plated onto Amp plate

Jeremiah & Chris:

Purified samples of 1 and 8 of SBP-TBP-VB12

1. 1 – 194ng/ul #2 – 239ng/ul

Samples 1 and 8 sent to NV Genomic Center for Sequencing

Purified LRP already digested (XbaI and PstI)

SBP-TBP-VB12 (TBP++) digested to ligate with LRP

Ligation of LRP à TBP++

Justin Emlen:

Transform the SBP-B12 insert in the expression plasmid into TOP 10 competent cells

June 29

Jasmine, Joe, and Michelle:

Checked 8 EP* colonies with colony PCR

Cultured colonies 2 and 6 in TB-Amp

Amplified RFP* and CP using PCR

Digested RFP* with SpeI, PstI, and DpnI

Jeremiah & Chris:

Transformation of LRP-TBP++

Phusion transformation of SBP-TBP à expression plasmid on LB-Amp

Justin and Dafne:

Colony PCR check SBP-B12 insert in expression plasmid

Three successful colonies were obtained

Culture colonies in 6 ml of TB-amp