Hylei at 22:27, 30 July 2011

2011-07-30T22:27:22Z

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= Excellgen PCR and PCR cloning Protocol =

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

 

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

 

== 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

 

=== PCR Cycle ===

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.

== Cloning of PCR Product ==

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. 

=== ===

=== Gel Purification ===

Cut out band from gel using green plastic cutter. 

Add 0.5 to 1 ml QG buffer. 

Dissolve gel at 50 oC. 

Cool, load to a silica gel spin column 

spin for 15 sec 

wash 2X with 300 ÎŒl PE 

spin 1 min at max speed 

use new autoclave tube 

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

 

=== Restriction Digestion ===

Add 

2.5 ÎŒl 10 X (fastdigest) buffer 

0.5 ÎŒl enzymes 

digest at 37 oC for 30 min 

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

=== ===

=== Column Purification ===

Add 120 ÎŒl PB buffer to digested PCR product 

add digested vector 

add mixture to a silica gel spin column 

spin for 15 sec 

wash 2X with 300 ÎŒl PE 

spin 1 min at max speed 

use new autoclave tube 

elute DNA with 20 ÎŒl water.

 

=== Ligation ===

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

 

=== Transformation ===

take 10 ÎŒl ligation mixture to 100 ÎŒl competent cells 

(leave the rest of 15 ÎŒl ligation reaction on bench for next day if no colony is grown on plate) 

incubate on ice for 30 min 

heat shock for 1 min to 1.5 min 

Chill on ice for 1 min 

add 350 ÎŒl SOC or LB medium 

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

 

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

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

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. 

[[Category:Protocols]]

PCR - Revision history

Hylei at 22:27, 30 July 2011