Objective
- Run analytical gel on ligations from 7/7/11 with negative control(s) and plasmid
- Do a QuikChange PCR on the ligation reaction to try to amplify the correct plasmid product prior to transformation. For now, we can use the his-tag primers that were used to make pTXB1His, but this will only work for the V(His)+I reaction. For the V+I reaction, we will have to design new primers (but should wait until we confirm that this approach will work.
- In the meantime, repeat the ligation where the V:I ratio was increased to minimize the self-ligation of the insert seen in the only ligation reactions which have shown any promise. (Although we don't know why the last time we ran this ligation it wouldn't have worked)
Bench work
- Analytical gel (0.8% agarose)
- (Besides 10μL ladder: 5μL sample + 1μL 0.3% bromophenol blue 20mM KPi)
- Lane 2: Ladder
- Lane 4: pTXB1.His+BSA ligation reaction from 7/7/11
- Lane 6: pTXB1+BSA ligation reaction from 7/7/11
- Lane 8: pTXB1+His (-) control from 6/29/11
- Lane 10: pTXB1 from 6/16/11
- QuikChange
- Ligation
- same procedure as 7/07/11 with same cut samples
- 8.5μL ddVHis + 4μL ddI + 2μL T4 ligase buffer + 4.5 μL sterile dH2O + 1μL T4 DNA ligase
- 8.5μL ddV + 4μL ddI + 2μL T4 ligase buffer + 4.5 μL sterile dH2O + 1μL T4 DNA ligase
- → 16°C O/N
- Replated 450μL of yesterday's transformations
- pTXB1.His+BSA in DH10B, etc.
- pTXB1+BSA in DH10B, etc.
Results
- Gel:
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AU CHEM-570 Lab Prep
