Team:Nevada/Week 4

June 11

Jeremiah & Chris:

Purified and digested samples of both #2 and #4 with XbaI and SpeI

Ran on gel to ensure existence of desired plasmid

Digestions on SBP, TBP, RFP, and SBP-TBP failed.

Ligated RFP à SBP

Ligated VB12 à SBP-TBP

SBP-TBP à expression vector

Jasmine:

Checked EP plate colonies using colony PCR

Grew cultures of colony #5 and #7

Michelle and Joe:

Check digestions: SBP + LRP (EcoRI and PstI) and PCR clean up/check of RFP (XbaI and PstI).

Ligate 6/6 expression digest: SBP (SpeI and PstI) and LRP (PstI and XbaI) digested with SpeI and XbaI with RFP backbone.

Digest RFP with XbaI and PstI.

Dephosphorylate SBP + LRP (SpeI and PstI).

Justin and Dafne:

Ligate SBP-B12 digest into Expression plasmid

Expression plasmid was digested and dephosphorylated by Jasmine

Ligation took place overnight

June 12

Michelle and Joe:

Redigest RFP with XbaI and PstI.

Transformation of ligation of 6/6 expression digest: SBP (SpeI and PstI) and LRP (PstI and XbaI) digested with SpeI and XbaI with RFP backbone into competent cells onto Ampicillin (AMP) plates.

Jeremiah & Chris:

Transformations of ligations from 6/11

SBP-TBP-VB12 --> LB + Kan

SBP-TBP-ExVector --> LB + Amp

SBP-RFP --> LB +Ka

Repeat of ligations from 6/11 – incubated overnight.

Justin and Dafne:

Digest SBP-B12 plasmid by Xba I and Spe I

For the creation of SBP-B12-SBP Plasmid

This project was not seen to its entirety

June 13

Jasmine:

Miniprepped EP cultures

Digested EP with SpeI

Digested RFP with XbaI and SpeI

Dephosphorylated digested EP

Ligated EP and RFP

Michelle and Joe:

Check transformation of 6/6 expression digest: SBP (SpeI and PstI) and LRP (PstI and XbaI) digested with SpeI and XbaI with RFP backbone into competent cells onto Ampicillin (AMP) plates.

Digest SBP + LRP with four enzymes: XbaI, EcoRI, PstI, and SpeI and PCR followed by PCR purification and PCR.

Jeremiah & Chris:

Transformations of ligations from 6/12

SBP-TBP-VB12 on LB + Ka

SBP-TBP-ExVector on LB + Amp

SBP-RFP on LB +Ka

Digested SBP-TBP again for use with expression vector with XbaI, SpeI, PstI, EcoRI

Justin and Dafne:

Ligate SBP-B12 insert (Xba I and Spe) into Expression plasmid

June 14

Jasmine:

Transformed RFP-EP ligation into competent cells

Plated transformation onto Amp plates

Michelle and Joe:

Ligation of digestion SBP + LRP (XbaI, EcoRI, PstI, and SpeI) from yesterday with RFP backbone using ligation protocol and Roche method.

PCR and PCR purify RFP before ligating using Invitrogen TA ligation kit followed by transformation.

Jeremiah & Chris:

PCR cleaned ligations from 6/13

Transformed SBP-TBP-ExV àLB+Amp (only one colony grew)

Digested SBP+TBP and ligated with expression vector using both standard and Roche methods. (Having problems getting SBP-TBP to express, hypothesis is that proteins in too high concentration are fatal to cells)

Justin and Dafne:

Transform expression plasmid with SBP-B12 insert

This transformation did not give positive results

June 15

Jasmine:

Checked RFP-EP plate

Michelle and Joe:

Check transformation of RFP ligation from yesterday. PCR colony check colonies from RFP

TA transformation plate and ran through gel to check colonies.

Tranformation of ligation SBP + LRP (XbaI, EcoRI, PstI, and SpeI) with RFP backbone onto AMP plates.

Jeremiah & Chris:

Colony PCR of single colony grown.

Transformation of Roche and standard ligations from 6/14 onto LB-Amp

Justin and Dafne:

Digest SBP-B12 plasmid with Xba I and Pst I HF

Ligate into new expression plasmid created by Jasmine