Problem with restriction digestion of cloned PCR product - (Dec/01/2015 )

Hi all,

We have used the forward and reverse primers for alpha amylase gene and the primers contain the following parts.

Forward primer: Xho1,Kex2 Cleavage site  and a part of alpha amylase coding region fro 5 to 3.

Reverse primer: part of alpha amylase gene, his tag sequence, stop codon and Xho1 restriction sequence.

The PCR product was cloned in to pGEMT easy vector. The vector was digested with Xho 1 restriction enzyme at 37 degrees for 2 hours. But the gel analysis showed the digestion has not worked. 

When we analysed the sequence the sequence of the cloned product (gene and the vector) using bioedit, the primer sequence of the his tag as well as the restriction site  did not properly align but the other parts properly aligned. 

Can any body tell what could be the reason for the change of the sequences?

Thanks 

Sounds like the primers were faulty - either in manufacture or by incorrect entering of the seqence.

Make sure that the primers all have the added bits on the 5' end especially the reverse... if you have them in the order written above, the tags etc will be on the 3' end!

Thanks for the comment! Here are the primer sequences in the 5 to 3 order. can any one tell us what could have gone wrong in the sequence? Thanks.....

CCG CTC GAG AAA AGA GAG GCT GAA GCT ATG TTC   CTG CTC GCG TTT TTG C -3’ (forward primer)

and  NNN NCT CGA GTT ATT AAT GAT GAT GAT GAT GAT GAT GAG GCC ATG CCA CCA ACC  ACC GTG G-3’ (reverse primer).

In reverse primer, GAT GAT GAT GAT GAT GAT - his tag

C^TCGA_G Xho 1 sequence

in forward primer, AAAAGA : Kex2 cleavage site

The primers are stuffed... the HIS tag is inverted - it needs to be the reverse complement of the coding sequence, not the complement. Currently you are coding for ILE...

Ohh thanks......but because it's a long primer will it affect the process? and can you run the primer in a PAGE and get the most intense fragment purified to get the CORRECT primer from a commercial primer solution? the analysed sequence does not have Xho 1 site.... might be an error in synthesis? 

You can't purify an incorrect primer and make it correct.