Team:Nevada/Week 11

July 30

Joe:

Transform SBP-RFP to EP-CP

Plate on AMP

TET/IPTG- miniprep of cultures

Dafne and Michelle:

Digest CP-EP* L-arabinose with SpeI and NSI).

Digest SBP (SpeI and PstI)-LRP (PstI and XbaI) with SpeI and EcoRI.

PCR purification of L-arabinose EP and ran gel.

Justin and Dafne

Removed dialysis membrane from 8M urea solution, placed in 6M urea solution

Run another expression to determine if there is a concentration of L-arabinose that could be used in order to :::induce protein expression without causing it to be moved to inclusion body

SDS-PAGE and Western blot analysis confirmed that SBP-B12 protein will be moved to inclusion body no matter the :::L-arabinose concentration

July 31

Joe:

SBP-RFP in EP-CP plates show no growth, PCR more

PCR purification - gel 120 bad

PCR more Ep-Cp

PCR purification - gel 121 compared before and after purification - Purification bad

PCR more Ep-Cp - gel 123 bad

Digestion ep-cp by Nsi/SpeI

Michelle:

Miniprep, PCR using sense primer (DNA2PTFsense) and antisense primer (DNA2PTFanti),

PCR purification, run sample with PCR purification and one without PCR purification through

gel, and digest SBP (SpeI and PstI)-LRP (PstI and XbaI) that was PCR purified with XbaI and PstI.

Cultured 2 colonies of CP-EP* with L-arabinose with TB-AMP.

Justin and Dafne

Place dialysis membrane in 4M Urea buffer

August 1

Joe:

Ep-Cp PCR, purify by glass fiber purification

Gel 125 shows good purification

Primer stocks- terminatior, tetrbs, lacrbs

PCR of each part - gel 125 good

Digest each by Nsi/SpeI

Michelle:

Run gel of yesterday’s digestion SBP (SpeI and PstI)-LRP (PstI and XbaI) with XbaI and PstI

and dephosphorylate.

Miniprep CP-EP* with L-arabinose, nanodrop, and digest with SpeI and NSI.

PCR SBP (SpeI and PstI)-LRP (PstI and XbaI) in preparation for PCR purification tomorrow

using glass milk.

Justin and Dafne:

Place dialysis membrane in 2M urea buffer

August 2

Joe:

Gel 126 shows all digestions successful

4cut digestion (EcoRI, XbaI, SpeI, PstI) of SBP-RFP

Ligations- 4cut with tetrbs/terminatior, 4cut with lacrbs/terminator,

Ep-Cp with SBP-RFP(XbaI/PstI)

Michelle:

PCR purification of SBP (SpeI and PstI)-LRP (PstI and XbaI) using glass milk.

Run gel of CP-EP* L-arabinose (SpeI and NSI) and dephosphorylate.

Ligate digestion of SBP (SpeI and PstI)-LRP (PstI and XbaI) with XbaI and PstI with CP-EP*

L-arabinose (SpeI and NSI.

August 3

Joe:

Transform ligations

PCR of ligations - gel 128 wrong sizes

Digestion of SBP-RFP by SpeI/PstI

Ligation of SBP-RFP(SpeI/PstI) to terminator

Michelle:

Transformation of ligation of SBP (SpeI and PstI)-LRP (PstI and XbaI) digested with XbaI and

PstI and CP-EP* L-arabinose (SpeI and NSI) onto AMP plate.

Justin and Dafne

Place dialysis membrane in pure water