cloning of gene in pcr4-topo vector or PQE 30 not successful

[nesma7]

I synthesized a gene of 192 bp coding for miniproinsulin by a gene synthesis company.

After recieving the gene I tried the cloning, but I faced a problem.
Here is a brief sequence of events since I started to use the gene:
- the gene was sent to me in pCR4-TOPO vector, I tried to propagate it in DH5α but the obtained plasmid after miniprep was shorter in size than expected & did not contain my restriction sites ( BamHI & HindIII ).
- I tried to propagate it in Top10 E. coli as it is known to give better results with TOPO plasmids, but I got the same result as with DH5α ( in fact the obtained plasmid was nearer to the expected size but it was still shorter than normal & didnot contain my restriction sites ).
- I checked the presence of my gene in pCR4-TOPO by PCR, I got a fragment of the proper size which upon digestion by BamHI & HindIII gave the gene having the correct size on agarose gel.
- I cloned the digested gene obtained by PCR in pQE-30 Xa ( Qiagen ) & checked the formed construct by PCR. The ligation was successful.
- Upon transforming the ligation product into DH5α I got no colonies ( regarding that a +ve control of pQE-30 Xa without my insert gave good transformation results ).
- I tried to propagate the pQE - gene construct in M15 ( Qiagen ) & Top10, but I always get no colonies in the test plate ( regarding that +ve & -ve control plates are good ).
that's all .............
Could any one help ....?


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[karthikmgt]

hi there...

did u add IPTG when you spread plated your transformation reaction mix.... bcos pCR4 TOPO has lac promoter that governs the entire vectors genes(amp,kan) and in dh5alpha lac repressor is active... so i think if you add IPTG and spread plate you will get colonies.....