20.109(S08): TA notes for module 1

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:

1. PCR Master Mix (2.5X), ~ 50 μL per group
2. DI water, prep 100 μL aliquot per group
3. 5 μL aliquots each of pCX-EGFP, D32N-fwd, D32N-rev
4. 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:

1. Qiagen QIAquick PCR purification kit
   • order # 28104, 50 rxns
   • 1 rxn per group
2. pCX-NNX, 10 μL per group
3. NEB2 Buffer (~5 μL used per group)
4. 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:

1. Qiagen QIAquick gel extraction kit
   • 2 extractions per group
   • order # 28704, 50 rxns
2. isopropanol (prep 500 μL aliquots)
3. sterile DI water (prep 500 μL aliquots)
4. loading dye for agarose gel electrophoresis (prep 35 μL aliquots)
5. 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
6. 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:

1. Retrieve at last minute and keep on cold rack: ligation buffer, T4 ligase
2. 3 M sodium acetate (prep 100 μL aliquots)
3. Yeast tRNA (prep 25 μL aliquots)
4. Cold 100% ethanol (prep 1 mL aliquots)
5. Cold 70% ethanol (prep 3 mL aliquots)
6. Sterile DI water (prep 100 μL aliquots)
7. pCX-EGFP (1 μL/50 ng per group)
8. competent cells
   • XL1-Blue from Stratagene, cat # ?200249?
   • 1 tube per group
9. LB+Amp plates, 5 per group + spares
   • VWR cat. #
   • may get ~ 40 plates/Liter LB
10. LB liquid medium

Day of Lab:

• Quiz (prepared by TA).
• Keep reagents for ligation reactions cold, make available to students as needed.
• Demonstrate and supervise bacterial transformation protocol.