Cloning protocol pLIC vectors

Summary

A protocol for ligation independent cloning.

Procedure

Design of PCR primers

This method provide a standard nucleotide sequence for the primers of PCR

5’ CCA GGG AGC AGC CTC G|---------------------| 3’

ORF

3’ |---------------------| ATT GCT CCG GCC ACG AAA CG 5’

Treatment of PCR products

After PCR, purify the product obtained and treat 10 µL in the following reaction:

10 µL PCR product

2 µL Buffer T4 DNA Polymerase

2 µL dATP (25 mM)

1 µL DTT (100 mM)

0,4 µL T4 DNA Polymerase

4,6 µL H2O

Incubate at room temperature for 30 minutes

Inactivate the reaction at 75°C for 20 minutes

The PCR product obtained and treated with TA DNA Poymerase looks like the following schema:

5’ CCA GGG AGC AGC CTC G |---------------------| TAA CGA 3’

ORF

3’ AG C|---------------------| ATT GCT CCG GCC ACG AAA CG 5’

Annealing mix

For the annealing mix, add 2 µL of PCR product, treated T4 DNA Polymerase and 1 µL of vector pre treated (make a negative control with 2µL of water instead of the insert).

Incubate at room temperature for 10 minutes

Add 1 µL of EDTA (25 mM)

Incubate at room temperature for 10 minutes

Transformation

The annealing mix can be transformed either in electro competent or in chemically competent cells. 2µL of the mix are transformed.

Further details

Protocol provided by Filip Glavan.

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