Griffitts:PCR: Difference between revisions

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** For amplifying rhizobium sequences, boil a dense suspension of cells in 35 μL [[Griffitts:Common buffers#colony lysis solution (for PCR) (100 mL)|PCR lysis buffer]] for ~2 min and vortex
** For amplifying rhizobium sequences, boil a dense suspension of cells in 35 μL [[Griffitts:Common buffers#colony lysis solution (for PCR) (100 mL)|PCR lysis buffer]] for ~2 min and vortex
** For amplifying ''E. coli'', adding cells directly to the reaction is sufficient
** For amplifying ''E. coli'', adding cells directly to the reaction is sufficient
* Thaw buffer, dNTPs and primers
* Thaw the ''Taq'' buffer, dNTPs, and primers
** Keep polymerase on ice throughout the procedure
** Keep ''Taq'' polymerase on ice throughout the procedure
* In standard PCR tube combine components according to the recipe below
* In standard PCR tube(s) combine components according to one of [[Griffitts:TaqPCR#Reaction recipes|the recipes below]]
** Set the reaction up on ice
** Set the reaction up on ice
** When running multiple samples, a Master Mix, which includes everything except the except the template and the primers (add 43.4 μL Master Mix to each tube)
** If performing multiple PCRs with the same primers then just use 100 μM stock, calculate for 0.3 μL per reaction, and correct the volume of water
* Select the appropriate cycling program and verify that all of the parameters are correct
* Select the appropriate cycling program and verify that all of the parameters are correct
** Allow 1 minute of extension time per 1 KB DNA being amplified
** Allow 1 minute of extension time per 1 KB DNA being amplified
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* Lower the lid until it latches and slightly tighten the heated lid onto the tubes
* Lower the lid until it latches and slightly tighten the heated lid onto the tubes


==Standard reaction recipe==
==Reaction recipes==
37 μl H<sub>2</sub>O<br>
===Standard recipe===
5 μl [[Griffitss:Stock_solutions#10X_Taq_buffer|10X ''Taq'' buffer]]<br>
Use this for 2 reactions
1 μl [[Griffitss:Stock_solutions#10_mM_DNTPs|dNTPs]]<br>
* 37 μL dH<sub>2</sub>O
0.4 μl ''Taq'' polymerase<br>
* 5 μL [[Griffitss:Stock_solutions#10X_Taq_buffer|10X ''Taq'' buffer]]
1 μl template<br>
* 1 μL [[Griffitss:Stock_solutions#10_mM_DNTPs|dNTPs]]
3 μl forward primer<br>
* 0.4 μL ''Taq'' polymerase<br>
3 μl reverse primer<br><br>
* 1 μL template
* 3 μL forward primer
* 3 μL reverse primer
Note: [[Griffitts:TaqPCR#Standard Primers|dilute the primers]] to 10X from the 100X stock in the freezer
 
===Master Mix (without primers)===
Use this for 3 to 4 reactions or reactions with multiple primer pairs<br>
Multiply by the number of reactions:
* 40 μL dH<sub>2</sub>O
* 5 μL [[Griffitss:Stock_solutions#10X_Taq_buffer|10X ''Taq'' buffer]]
* 1 μL [[Griffitss:Stock_solutions#10_mM_DNTPs|dNTPs]]
* 0.4 μL ''Taq'' polymerase
Add 46.4 μL to each PCR tube and use a [[Griffitts:TaqPCR#Primer Master Mix|Primer Master Mix]]<br>
 
===Master Mix (with primers)===
Use this for 5 or more reactions with the same primer pairs<br>
Multiply by the number of reactions:
* 42.4 μL dH<sub>2</sub>O
* 5 μL [[Griffitss:Stock_solutions#10X_Taq_buffer|10X ''Taq'' buffer]]
* 1 μL [[Griffitss:Stock_solutions#10_mM_DNTPs|dNTPs]]
* 0.4 μL ''Taq'' polymerase
* 0.3 μL forward primer
* 0.3 μL reverse primer
Note: Use the 100X stock in the freezer (''don't'' dilute)<br>
Add 49.4 μL to each PCR tube
 
==Primer Dilution==
===Standard Primers===
* 27 μL dH<sub>2</sub>O
* 3 μL primer
Repeat for both primers
 
===Primer Master Mix===
* 24 μL dH<sub>2</sub>O
* 3 μL forward primer
* 3 μL reverse primer

Revision as of 11:20, 13 February 2008


PCR with Taq Polymerase


Procedure

  • Prepare your template sample
    • For amplifying rhizobium sequences, boil a dense suspension of cells in 35 μL PCR lysis buffer for ~2 min and vortex
    • For amplifying E. coli, adding cells directly to the reaction is sufficient
  • Thaw the Taq buffer, dNTPs, and primers
    • Keep Taq polymerase on ice throughout the procedure
  • In standard PCR tube(s) combine components according to one of the recipes below
    • Set the reaction up on ice
  • Select the appropriate cycling program and verify that all of the parameters are correct
    • Allow 1 minute of extension time per 1 KB DNA being amplified
  • Select "Run program" and select "YES" when asked about heated lid
  • Wait until block has reached the beginning temperature. Place PCR tube into the machine
  • Lower the lid until it latches and slightly tighten the heated lid onto the tubes

Reaction recipes

Standard recipe

Use this for 2 reactions

  • 37 μL dH2O
  • 5 μL 10X Taq buffer
  • 1 μL dNTPs
  • 0.4 μL Taq polymerase
  • 1 μL template
  • 3 μL forward primer
  • 3 μL reverse primer

Note: dilute the primers to 10X from the 100X stock in the freezer

Master Mix (without primers)

Use this for 3 to 4 reactions or reactions with multiple primer pairs
Multiply by the number of reactions:

Add 46.4 μL to each PCR tube and use a Primer Master Mix

Master Mix (with primers)

Use this for 5 or more reactions with the same primer pairs
Multiply by the number of reactions:

  • 42.4 μL dH2O
  • 5 μL 10X Taq buffer
  • 1 μL dNTPs
  • 0.4 μL Taq polymerase
  • 0.3 μL forward primer
  • 0.3 μL reverse primer

Note: Use the 100X stock in the freezer (don't dilute)
Add 49.4 μL to each PCR tube

Primer Dilution

Standard Primers

  • 27 μL dH2O
  • 3 μL primer

Repeat for both primers

Primer Master Mix

  • 24 μL dH2O
  • 3 μL forward primer
  • 3 μL reverse primer
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