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PCR standard - Same primers

Category: Experimental Procedures
Author: Admin eLabJournal

Labels: Cloning, DNA

Materials

 

- PCR reaction buffer (10X)

- dNTPs (10X)

- MgCl2  ( 25--other--mM )

- DMSO (100%)

- TaqTaq Hi-FiExpandPfuPfu Turbo--other-- polymerase  ( 10--other-- U µl-1)

- Template

- Primers

- PCR machine

Experiment settings

Number of samples	123451020--other--
Reaction volume:	2550--other--µl	make 0510--other--%  more
Template Type:	genomic DNAplasmid--other--
Template Name:	E. coli--other--	0.10.20.51--other--µl /reaction
Primer 1:	forward primer--other--	0.10.20.51--other--µl /reaction
Primer 2:	reverse primer--other--	--other--µl /reaction

Step 1

Prepare a the following reaction mix:
 

PCR reaction buffer (10X)	--other--µl
dNTP mix (10X)	--other--µl
02.55--other--mM  MgCl2  =	--other--µl
0510--other--%  DMSO  =	--other--µl
TaqTaq Hi-FiExpandPfuPfu Turbo--other--  polymerase (--other--  U µl-1)	--other--µl
Primer 1: forward primer--other--	--other--µl
Primer 2: reverse primer--other--	--other--µl
dH20	--other--µl
Total	--other--µl

Step 2

Vortex the reaction mixture and add --other--µl  to 123451020--other--  PCR tubes

Step 3

Add template to each tube:

--other--µl

--other-- --other--

Step 4

Mix the tubes, spin down briefly and program the PCR machine to start the reaction. After PCR proceed to DNA agarose gel electrophoresis

• Keep the reaction mix on ice as much as possible, especially after the polymerase enzyme is added!
   

 

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