Difference between revisions of "20.109(S08): TA notes for module 1"
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#*pCX-NNX, EcoRI cut only | #*pCX-NNX, EcoRI cut only | ||
#*large (400 μL) and small (25 μL) volume reactions should be done for each of the above | #*large (400 μL) and small (25 μL) volume reactions should be done for each of the above | ||
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'''Day of Lab:''' | '''Day of Lab:''' | ||
| Line 78: | Line 79: | ||
*Run purified products (2 per group) on an agarose gel at the end of the day - post photograph. | *Run purified products (2 per group) on an agarose gel at the end of the day - post photograph. | ||
*Freeze DNA at end of day. | *Freeze DNA at end of day. | ||
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| + | ===[[20.109%28S08%29:DNA_engineering/DNA_ligation_and_bacterial_transformation_%28Day_4%29 | Day 4]]=== | ||
| + | |||
| + | '''Materials required:''' | ||
| + | |||
| + | # Retrieve at last minute and keep on cold rack: ligation buffer, T4 ligase | ||
| + | # 3 M sodium acetate (prep 100 μL aliquots) | ||
| + | # Yeast tRNA (prep 25 μL aliquots) | ||
| + | # Cold 100% ethanol (prep 1 mL aliquots) | ||
| + | # Cold 70% ethanol (prep 3 mL aliquots) | ||
| + | # Sterile DI water (prep 100 μL aliquots) | ||
| + | # pCX-EGFP (1 μL/50 ng per group) | ||
| + | # competent cells | ||
| + | #* XL1-Blue from Stratagene, cat # ?200249? | ||
| + | #* 1 tube per group | ||
| + | # LB+Amp plates, 5 per group + spares | ||
| + | # LB liquid medium | ||
| + | |||
| + | '''Day of Lab:''' | ||
Revision as of 21:56, 27 December 2007
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 PCR purification 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.
Day 3
Materials required:
- Qiagen QIAquick gel extraction kit
- 2 extractions per group
- order # 28704, 50 rxns
- isopropanol (prep 500 μL aliquots)
- sterile DI water (prep 500 μL aliquots)
- loading dye for agarose gel electrophoresis (prep 35 μL aliquots)
- 1% agarose gels prepared in 0.5X TBE buffer
- each group has 10 samples, so requires 10 wells
- prepare 1 gel per two groups, but with 2 combs
- also prepare 1 more gel (2 combs!), for running purified products
- Single-enzyme digests - may be prepared on Day 2.
- pCX-NNX, XbaI cut only
- pCX-NNX, EcoRI cut only
- large (400 μL) and small (25 μL) volume reactions should be done for each of the above
Day of Lab:
- Quiz (prepared by TA).
- Run purified products (2 per group) on an agarose gel at the end of the day - post photograph.
- Freeze DNA at end of day.
Day 4
Materials required:
- Retrieve at last minute and keep on cold rack: ligation buffer, T4 ligase
- 3 M sodium acetate (prep 100 μL aliquots)
- Yeast tRNA (prep 25 μL aliquots)
- Cold 100% ethanol (prep 1 mL aliquots)
- Cold 70% ethanol (prep 3 mL aliquots)
- Sterile DI water (prep 100 μL aliquots)
- pCX-EGFP (1 μL/50 ng per group)
- competent cells
- XL1-Blue from Stratagene, cat # ?200249?
- 1 tube per group
- LB+Amp plates, 5 per group + spares
- LB liquid medium
Day of Lab:
