Editing PCR

= Excellgen PCR and PCR cloning Protocol  =

'''Notes: Tag for screening, Pfu or Phusion for cloning. ''' 

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$2500 deduction will be made from your next paycheck if you containminate primers, template or PCR enzymes, or use Pfu or Phusion for screening 

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== PCR  ==

=== Make PCR mix  ===

if you want to run 10 PCR, make mix for 11 PCR reactions 

for each reaction: 

2.5 ÎŒl of 10X PCR buffer 

0.5 or 1 ÎŒl 10 mM dNTP 

0.5 or 1 ÎŒl F primer 

0.5 or 1 ÎŒl R primer 

MgSO4 or MgCl2 if 10X PCR buffer does not contain Mg2+ 

some template. 

0.5 or 1 unit Taq, Pfu or Phusion, use long tip. 

water to 25 ÎŒl 

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=== PCR Cycle<br>  ===

step 1: 95 oC 

step 2: 95 oC 

step 3: 55 oC 

step 4: 68 or 72 oc for 1min per kb 

back to step 2 for 25 or 30 cycles. for gene synthesis max 25 cycles 

step 5: 72 oC for 5 min 

no 4 oC incubation 

===  ===

=== Agarose Gel  ===

Run 5 to 10 ÎŒl PCR on 1% agarose gel, 0.5X TAE, 200 v, 15 to 30 min. 

== <br>Cloning of PCR Product<br>  ==

If PCR product is to be used for cloning, load all PCR product to gel. Cut out desired band or bands, gel purify. 

Time required: 1 full day is required from PCR to transformation.<br>

===  ===

=== Gel Purification  ===

Cut out band from gel using green plastic cutter. <br> 

Add 0.5 to 1 ml QG buffer. <br> 

Dissolve gel at 50 oC. <br> 

Cool, load to a silica gel spin column<br> 

spin for 15 sec <br> 

wash 2X with 300 ÎŒl PE<br> 

spin 1 min at max speed<br> 

use new autoclave tube<br> 

add 20 ÎŒl H2O, elute DNA by spin at max speed for 1 min. 

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=== Restriction Digestion  ===

Add <br> 

2.5 ÎŒl 10 X (fastdigest) buffer<br> 

0.5 ÎŒl enzymes<br> 

digest at 37 oC for 30 min<br> 

For NEB buffer, 1 hour to 2 hours are required. 

=== <br>  ===

=== Column Purification  ===

Add 120 ÎŒl PB buffer to digested PCR product<br> 

add digested vector<br> 

add mixture to a silica gel spin column<br> 

spin for 15 sec<br> 

wash 2X with 300 ÎŒl PE<br> 

spin 1 min at max speed<br> 

use new autoclave tube<br> 

elute DNA with 20 ÎŒl water. 

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=== Ligation  ===

Add 2.5 ÎŒl 10X ligase buffer, add 1 ÎŒl or 0.5 ÎŒl ligase, ligate at RT for 2 hours. 

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=== Transformation  ===

take 10 ÎŒl ligation mixture to 100 ÎŒl competent cells<br>

(leave the rest of 15&nbsp;ÎŒl ligation reaction on bench for next day if no colony is grown on plate)<br> 

incubate on ice for 30 min<br> 

heat shock for 1 min to 1.5 min<br> 

Chill on ice for 1 min<br> 

add 350 ÎŒl SOC or LB medium<br> 

put into an eppendorf shaker to incubate for 30 min (Amp) to 1 hour (Kan), 750 rpm. 

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Spin down bacteria cells, pour off medium (leave about 100 ÎŒl medium)<br> 

resuspend cells using pipette, add all cells to a LB plate with antibiotics<br> 

spread cells evenly on plate using glass beads

incubate at 37 oC O/N (agar side up). 

=== Documentation  ===

Take a picture, upload result to ELN. 

$800 will be deducted from your paycheck if you use a razor blade to cut out DNA bands.<br> 

[[Category:Protocols]]