Team:Alberta/Notebook/protocols/pcr

From 2010.igem.org

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==General Protocols==
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-----------------------------
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*[[Team:Alberta/Notebook/protocols/invitro_biobyte_assembly | In Vitro BioByte Assembly]]
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-----------------------------
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*[[Team:Alberta/Notebook/protocols/LB | LB Plates and Broth]]
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*[[Team:Alberta/Notebook/protocols/transformations |Transformations]]
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*[[Team:Alberta/Notebook/protocols/overnight |5mL Overnight ]]
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*[[Team:Alberta/Notebook/protocols/glycerol | Glycerol Stock ]]
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-----------------------------
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*[[Team:Alberta/Notebook/protocols/miniprep | Plasmid Miniprep ]]
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*[[Team:Alberta/Notebook/protocols/digest | Restriction Digest ]]
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*[[Team:Alberta/Notebook/protocols/vector_dephos | Vector Dephosphorylation]]
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*[[Team:Alberta/Notebook/protocols/ligation | Ligation ]]
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-----------------------------
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*[[Team:Alberta/Notebook/protocols/agarose_gel | Agarose Gel Electrophoresis ]]
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*[[Team:Alberta/Notebook/protocols/gel_extraction | Gel Extraction ]]
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-----------------------------
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*[[Team:Alberta/Notebook/protocols/pcr | PCR]]
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*[[Team:Alberta/Notebook/protocols/colony_pcr | Colony PCR ]]
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*[[Team:Alberta/Notebook/protocols/pcr_purification | PCR Purification ]]
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-----------------------------
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*[[Team:Alberta/Notebook/protocols/labelling | Sample Labelling Conventions]]
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*[[Team:Alberta/Notebook/protocols/sequencing | Fluorescent Sequencing Reaction]]
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*[[Team:Alberta/Notebook/protocols/primer_design | Primer Design]]
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-----------------------------
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Latest revision as of 02:30, 27 October 2010

TEAM ALBERTA


PCR

Procedure:

  • Before you start:
    • KEEP EVERYTHING ON ICE. Put Taq back into freezer as soon as you`re done with it. DON`T put back dNTP tubes.
    • Reserve a thermocycler and check what size of tubes it takes.
    • Making a master mix conserves expensive reagents, so try to always use one.
    • You may have to do dilutions of your reagents in order to make them usable for PCR (ex: primers, plasmid DNA...)
    • DON'T PUT PRIMERS OR TEMPLATE INTO THE MASTER MIX. ADD POLYMERASE TO THE MASTER MIX LAST (after your other tubes already have template DNA and primers in them)!
  • To add into a tube:
PCR buffer 5ul
10uM dNTPs 1ul
50uM MgCl2 2ul
Forward primer 2.5ul
Reverse primer 2.5ul
1ng Template 1ul
Taq polymerase 0.5ul
MilliQ water 35.5ul
TOTAL 50ul
  • Program to run:
1. 94oC 3 minutes
2. 94oC 45 seconds
3. 62oC 30 seconds
4. 72oC 90 seconds
5. Cycle steps 1 to 4 "30" times
6. 72oC 10 minutes


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