Difference between revisions of "20.109(S08): TA notes for module 1"

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(→‎General notes)
(→‎Daily Notes)
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'''Materials required:'''  
 
'''Materials required:'''  
  
−
#Qiagen QIAquick kit  
+
#Qiagen QIAquick PCR purification kit  
 
#*order # 28104, 50 rxns
 
#*order # 28104, 50 rxns
 
#*1 rxn per group
 
#*1 rxn per group
Line 54: Line 54:
 
*Quiz (prepared by TA).
 
*Quiz (prepared by TA).
 
*Thaw PCR products and template on ice.
 
*Thaw PCR products and template on ice.
 +
 +
===[[20.109%28S08%29:DNA_engineering/Agarose_gel_electrophoresis_%28Day_3%29 | 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
 +
# 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, for each single cut
 +
 +
'''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.

Revision as of 21:26, 27 December 2007


20.109(S08): Laboratory Fundamentals of Biological Engineering

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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
  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, for each single cut

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.
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