Muratore:Protocols/PCR/Basic: Difference between revisions
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(New page: {|border="1" |+ Basic PCR protocol |- !Tube !!sterile H<sub>2</sub>O !!Pfu Buffer !!Template !!5'primer !!3'primer !!dNTPs !!Pfu Turbo !!wax |- ! !! !!(10X) !!(10 μg/mL) !!(4 μM) !!(4 μ...) |
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Basic PCR protocol | '''Basic PCR protocol''' | ||
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!Tube !!sterile H<sub>2</sub>O !!Pfu Buffer !!Template !!5'primer !!3'primer !!dNTPs !!Pfu Turbo !!wax | !Tube !!sterile H<sub>2</sub>O !!Pfu Buffer !!Template !!5'primer !!3'primer !!dNTPs !!Pfu Turbo !!wax | ||
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! !! !!(10X) !!(10 μg/mL) !!(4 μM) !!(4 μM) !!(10 mM ea) !!(2.5 U/μL) !! | ! !! !!(10X) !!(10 μg/mL) !!(4 μM) !!(4 μM) !!(10 mM ea) !!(2.5 U/μL) !! | ||
|- | |- | ||
! | !Experimental | ||
|72 μL ||10 μL ||5 μL ||5 μL ||5 μL ||2 μL ||1 μL ||50 μL | |72 μL ||10 μL ||5 μL ||5 μL ||5 μL ||2 μL ||1 μL ||50 μL | ||
|- | |- | ||
! | !(-) Control | ||
|73 μL ||10 μL ||5 μL ||5 μL ||5 μL ||2 μL ||0 μL ||50 μL | |73 μL ||10 μL ||5 μL ||5 μL ||5 μL ||2 μL ||0 μL ||50 μL | ||
|} | |} | ||
Revision as of 10:42, 26 May 2011
| Tube | sterile H2O | Pfu Buffer | Template | 5'primer | 3'primer | dNTPs | Pfu Turbo | wax |
|---|---|---|---|---|---|---|---|---|
| (10X) | (10 μg/mL) | (4 μM) | (4 μM) | (10 mM ea) | (2.5 U/μL) | |||
| Experimental | 72 μL | 10 μL | 5 μL | 5 μL | 5 μL | 2 μL | 1 μL | 50 μL |
| (-) Control | 73 μL | 10 μL | 5 μL | 5 μL | 5 μL | 2 μL | 0 μL | 50 μL |
- Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.
- 5' @ 95°C
- 30" @ 95°C
- 2' @ Tm-5°C
- 2' @ 78°C
- repeat steps 2-4 x 29 more times
- 5' @ 78°C
