Oct 18, 2020

Public workspacePCR

DOI
dx.doi.org/10.17504/protocols.io.bniemcbe
  • 1South China University of Technology
Jiaxin Li
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DOI: dx.doi.org/10.17504/protocols.io.bniemcbe
Protocol Citation: Jiaxin Li 2020. PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.bniemcbe
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 16, 2020
Last Modified: October 18, 2020
Protocol Integer ID: 43302
Set up a small box with ice, put DNA and 2×high Taq Master Mix into it before going into the Bio-rad S1000TM thermocycler cycler.
Add the following reagent to a PCR tube (50 μl) and program the thermocycler as follows:
Q5 polymerase
Q5 polymerase
component25ul50ul
5XQ5 buffer5ul10ul
10mM dNTPs0.5ul1ul
Forward Primer 10μM 1.25ul2.5ul
Reverse Primer 10μM 1.25ul2.5ul
Template (<1000ng)According to different templateAccording to different template
Q5 polymerase0.25ul0.5ul
ddWaterAdd to 25ulAdd to 50ul

Temperaturetime
98℃ 03:00
98℃00:05-00:10
Tm-5℃00:10-00:30
72℃20-30s/kb
72℃02:00
4℃∞
KOD FX
KOD FX
componentvolumn/ulFinal Concentration
2xPCR buffer25uL1X
2mM dNTPs10uL400umol/L
10umol/L Forward Primer1.5uL0.3umol/L
10umol/L Reverse Primer1.5uL0.3umol/L
Template DNA2uLGenomic DNA≤200ng/50ul
Plasmid DNA（10ng/ul）
cDNA≤200ng
Crude sample≤0.5-4ul
ddH2OAdd to 50uL（10ul）
KOD FX1uL1unit/50μl
Total50μl
Temperaturetime
98℃T1= 03:00
98℃T2= 00:10
Tm-5℃T3=00:30
68℃T4 : 1min/kb
68℃T6>T2+T3+T4
4℃∞
T5 Taq Polymerase
T5 Taq Polymerase
ComponentVolume/ulFinal Concentration
2XT5 Super PCR Mix5ul1x
10uM Forward Primer0.4ul0.4uM
10uM Reverse Primer0.4ul0.4uM
Template DNA1ul<1ug
ddH2OAdd to 10ul
Total10ul
Temperaturetime
98℃03:00
98℃00:10
Tm-5℃00:10
72℃20s/kb
72℃02:00
4℃∞