Common cloning kit experiments in common problems and solutions |

Test - lowercase jpg

Kaixin micro test

Test probe P100-M3

Common problems and solutions in seamless cloning kit experiments |

one,

No cloning or cloning on the plate

1,

possible reason

:

Primer sequence is incorrect

solution:

It was confirmed that the primer sequence contained a 15 bp homologous sequence identical to the vector insertion region.

2,

possible reason

:

PCR product is not pure

solution:

Optimize the PCR amplification reaction to obtain a single PCR product

;

Purify your PCR product with a different purification method.

3,

possible reason

:

The DNA concentration in the reaction system is too low

solution:

In the recombination reaction, the recommended amount of DNA is added.

4,

possible reason

:

Impure vector or insert inhibits recombination

solution:

Recombination reactions are recommended after purification of the DNA by gel recovery or spin column.

5,

possible reason

:

Too much recombination reaction solution added during the conversion process

solution:

The transformation volume of the recombinant reaction solution should not exceed 1/10 of the volume of the transformed cells.

6,

possible reason

:

Competent cell transformation efficiency is low

solution:

Replace fresh, efficient competent cells.

7,

possible reason

:

Adding wrong or excessive antibiotics to the medium

solution:

Add the right amount of antibiotics to the transformation plate.

two,

More false positive clones

1,

possible reason

:

Cloning vector linearization is incomplete

solution:

Re-cut your vector, increase the digestion time, and recover the gel.

2,

possible reason

:

The template plasmid used for PCR is resistant to the same resistance as the desired cloning vector.

solution:

Linearize the template plasmid before the PCR reaction

;

The PCR product was treated with DpnI, and the template plasmid was digested and then purified.

3,

possible reason

:

Long-term placement of the conversion plate leads to resistance failure

solution:

Confirm that the plate is freshly configured and contains the correct, appropriate amount of antibiotics.

three,

Clone contains incorrect insert

possible reason

:

PCR product mixed with non-specific fragments

solution:

Optimize the PCR amplification system to improve specificity or gel recovery and purification of the desired fragment.