1. Principles

Design a pair of primers that anneal to opposite strands of the regions flanking the one to be deleted. The primers have to be designed such that the subsequent ligation will not result in a frame-shift, i.e. the deletion has to be in frame. One of the primers should be phosphorylated. Both primers should at least be cartridge purified.

Primer A

5----------->3

5---------------------------------------------------------------------3

3---------------------------------------------------------------------5

3<--------------5

Primer B <----deletion-->

Take template DNA, and do a PCR with PFU using primers A+B. DpnI treat to digest the parental template DNA (DpnI will only cleave methylated DNA and methylation requires enzymes present in the bugs from which the DNA has been prepared but not present in your PCR-reaction), then stick that over a Qiagen PCR prep column. Add T4 ligase buffer and ligase, then transform some bugs. (Use one phosphorylated primer for an easy life, avoids having to kinase the pcr product.)

2. PFU-based deletion mutagenesis

The following protocol is based in parts on the quickchange mutagenesis protocol (Stratagene) which strictly speaking is not a true PCR.

With the Quickchange protocol for point mutations the primers are complementary to each other and will only anneal to the original template and not to the "new" DNA-strands. Hence quickchange should require higher amounts of DNA than a general PCR. This results in two complementary nicked strands of DNA. As the nicks are not at the same site, the "PCR" product can be transformed straight into bugs.

In contrast the deletion mutagenesis described here is a true PCR reaction; i.e. each primer will anneal to the complementary newly synthesised PCR-product primed by the other primer. This results in two linear complementary strands of DNA which have to be ligated prior to transformation into bugs. Stratagene have a protocol for this on the web (see Exsite.pdf). However they use a special polymerase mix which contains Taq and therefore is faster than PFU and use unnecessary large amounts of template.

3. Protocol for PFU-based deletion mutagenesis

Primers for Mutagenesis:

Should be >20 bp, have a Tm of 60°C or higher. anneal to opposite strands, face away from the deletion and be in frame. One of them should be phosphorylated and both should be at least cartridge purified. Concentration: 100 ng/µl

Template: as small as possible. Concentration: 10 ng/µl

Control tubes should be set up in parallel which should contain all of the components, except for Pfu polymerase. DpnI digestion will therefore destroy the template in these tubes. These tubes should be processed in parallel with the experimental tubes and act as negative controls at the transformation stage. A PCR-raection without template is another useful negative control.

Cycling parameters:

#4 1x 95°C 30 seconds

#1 13x 95°C 30 seconds

55°C 1 min

68°C 8 min (i.e. 1-2 min/kb)

2 min on ice

Add 1 µl DpnI (10 U) to each amplification reaction

Pipette up and down, spin down for 1 min and incubate at 37°C for 1 hour

Use PCR-elution kit to clean up the digest, elute in 30 µl

Ligation:

DNA 4 µl

Rapid T4-Liagsebuffer 5 µl

T4-Ligase 1 µl

Incubate 1 hour at RT (and 1 hr at 37°C)

Transform with 2 µl ligated PCR-reaction into 50 µl XL1blue supercompetent and plate the whole lot.

Set up a PCR reaction containing:-

1. Principles

This is an alternative classical protocol which will work.

FUP FDP

5---à 3 5------>3

5-------------------------------------------------------------------------------3

3-------------------------------------------------------------------------------5

3<-------5 3ß ----5

RDP RUP

FUP : Forward universal primer containing a unique restriction site RE1

RDP : Reverse deletion primer

FDP : Forward deletion primer

RUP : Reverse universal primer containing a unique restriction site RE2

PCR reactions :

PCR 1A : FUP/RDP : P1

PCR 1B : FDP/RUP : P2

P1 P2

Use the qiagen PCR-elution kit and run the two eluted PCR-products on a gel

Cut out the two bands separately and estimate amounts on gel.

Use 10-50 ng P1 and P2 for the fusion PCR reaction.

Fusion PCR reaction :

P1/P2 + FUP/RUP : FP1

Digests:

FP1 x RE1/RE2 => RF1

Original template x RE1/RE2 => RF2

Ligation 1 :

Ligate RF1 in RF2

Transformation :

Transform ligation 1 to an appropriate E.coli strain.