Barrick Lab :: ProtocolsQuickChange

---+ Site-directed mutagenesis protocol (adapted from QuickChange)
A protocol for changing one (or a few) bases on a plasmid

---++ SUPPLIES:

---+++ Primer Design:
Use the following website to design the QuickChange primers: </br>
http://www.bioinformatics.org/primerx/cgi-bin/DNA_1.cgi

---+++ Equipment:

   * Thermocycler
   * 37°C Incubator

---+++ Consumables:

   * PCR tubes
   * Reagents in table below
   * Dpn1 restriction enzyme (to remove template from rnx)

---+++ Buffers and Solutions:

<b>QuickChange rxn: </b>
| *Reagent* | *Amount / L* | 
| DNA template (~5ng/µl) | 2.0 µl  | 
| 5x HF buffer |  10 µl   |  
| 10 mM dNTPs |  1 µl   |  
| 10µM Forward Primer |  2.5 µl   | 
| 10µM Forward Primer |  2.5 µl   | 
| 100% DMSO |  2.5 µl   | 
| 50 mM MgCl<sub>2</sub> |  1 µl   | 
| Phusion Polymerase |  0.5 µl   | 
| H<sub>2</sub>O | 28 µl | 
| <b>TOTAL</b>| <b>50 µl</b> |


---++ PROTOCOL
Thermocycler settings:
   1) 98C 30 sec 
   2) 98C 30 sec 
   3) 55C 1 min (can be empirically optimized)
   4) 72F 1 min/kb (can change time for your polymerase of choice)
      * repeat steps 2-4 x12-18 
   5) hold at 10C

add 1µl Dpn1 to each tube and mix thoroughly. Incubate for 1hr at 37C (thermocycler or incubator). Proceed to transformation or store at -20C. 

-- Main.MattMcGuffie - 25 Oct 2018

Barrick Lab > ProtocolList > ProtocolsQuickChange

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Topic revision: r1 - 2018-10-25 - 20:12:41 - Main.MattMcGuffie