20.109(S08): TA notes for module 1
From Course Wiki
Revision as of 20:07, 27 December 2007 by AgiStachowiak (Talk)
Contents
General notes
Plasmids used:
- pCX-EGFP:
- contains full-length EGFP gene
- Amp resistant
- f1 ori
- SV40 ori
- pCX-NNX:
- a mock version of pCX-EGFP, contains no EGFP gene
- Amp resistant
- f1 ori
- some altered restriction sites compared to pCX-EGFP, and SV40 ori deleted
Key preparation:
- In case something goes wrong, spare PCR product should be prepared by TA prior to term.
Daily Notes
Day 1
Materials required:
- PCR Master Mix (2.5X), ~ 50 μL per group
- DI water, prep 100 μL aliquot per group
- 5 μL aliquots each of pCX-EGFP, D32N-fwd, D32N-rev
- 2 PCR tubes per group
Keep everything cold!
Day of Lab:
- Lab orientation quiz (not a TA quiz) will be taken today.
- Remember to freeze PCR products when they are ready.
Day 2
Materials required:
- Qiagen QIAquick kit
- order # 28104, 50 rxns
- 1 rxn per group
- pCX-NNX, 10 μL per group
- NEB2 Buffer (~5 μL used per group)
- EcoRI and XbaI enzymes (~2 μL used per group)
Day of Lab:
- Quiz (prepared by TA).
- Thaw PCR products and template on ice.