Team:Alberta/Notebook/protocols/pcr

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==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. 94<sup>o</sup>C || 3 minutes
|-
|2. 94<sup>o</sup>C || 45 seconds
|-
|3. 62<sup>o</sup>C || 30 seconds
|-
|4. 72<sup>o</sup>C || 90 seconds
|-
|5. Cycle steps 1 to 4 "30" times
|-
|6. 72<sup>o</sup>C || 10 minutes
|}



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