Difference between revisions of "Template:Team:Groningen/CONTENT/PROTOCOLS/PCR"
(Created page with " {{Team:Groningen/TEMPLATES/STEP |title=PCR mix |content=<html> <div class="text">1. Create the following mix.</div> <div class="object data" id="tbl1"> <div class="wrapper"> ...") |
|||
| Line 1: | Line 1: | ||
| − | |||
{{Team:Groningen/TEMPLATES/STEP | {{Team:Groningen/TEMPLATES/STEP | ||
|title=PCR mix | |title=PCR mix | ||
| Line 9: | Line 8: | ||
<div class="field fw3">Ingredient</div> | <div class="field fw3">Ingredient</div> | ||
<div class="field fw3">20 μl reaction</div> | <div class="field fw3">20 μl reaction</div> | ||
| − | <div class="field fw7">Final Concentration/div> | + | <div class="field fw7">Final Concentration</div> |
</div> | </div> | ||
<div class="record"> | <div class="record"> | ||
| Line 56: | Line 55: | ||
<div class="field fw3">Cycle Step</div> | <div class="field fw3">Cycle Step</div> | ||
<div class="field fw3">Temperature</div> | <div class="field fw3">Temperature</div> | ||
| − | <div class="field fw7">Time/div> | + | <div class="field fw7">Time</div> |
| − | <div class="field fw7">Cycle/div> | + | <div class="field fw7">Cycle</div> |
</div> | </div> | ||
Revision as of 21:08, 21 August 2015
{{Team:Groningen/TEMPLATES/STEP |title=PCR mix |content=
1. Create the following mix.
Ingredient
20 μl reaction
Final Concentration
\(H_2O\)
Add to 20 μl
5x Phusion HF buffer*
4 μl
1x
10 mM dNTPs
0.4 μl
200 μM each
Forward primer**
X μl
0.5 μM
Template DNA
X μl
(DMSO***, optional)
(0.6 μl)
(3%)
Phusion DNA polymerase
0.2 μl
0.02 U/ μL
PCR Mix.
2. Gently vortex the samples and spin down.
3. Place the reactions in a thermal cycler. Perform PCR using recommended thermal cycling conditions.
Cycle Step
Temperature
Time
Cycle
Initial denaturation
98°C
30 s
1
Denaturation
98°C
5-10 s
25-35
Annealing
T m - 5°C
10-30 s
25-35
Extension
72°C
15-30 s/kb
25-35
Final extension
72°C
5-10 min
1
Hold
4°C
Hold
1
Thermal Cycling.