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Why the insert become shorter after inserting into plasmid?


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#1 le_duy211

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Posted 24 February 2015 - 11:03 PM

Hi everyone!

I am having trouble with cloning. My PCR product is 1.7kb with EcoRI and SalI site at both ends. After ligation into the plasmid, I transformed into E. coli DH5alpha cell. The colonies growth slowly. I did the colony PCR for searching the candidate with the Forward primer which is anneal to the middle of the insert and the reverse primer which is anneal to the vector and I got the candidate colony. I purified the plasmid from these candidates and did restriction enzyme with the same enzyme for checking. But the insert from the digestion is 1.5kb, shorter than before ligation. Do you have any ideas about this situation? I did it for three times and the results were the same. What should I do next?

 

Thank in advance.

 



#2 bob1

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Posted 25 February 2015 - 12:20 AM

There are a few options here, the most likely of which is that you have an internal restriction site (or perhaps star activity), which is causing a shorter product. It is also possible but much less likely that you have a DNA that is forming some sort of secondary structure (hairpin?). A third option is that you have an internal amplification site for your primers, and what you cloned isn't what you thought it was.

 

Sequence it to find out.



#3 labtastic

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Posted 14 March 2015 - 11:49 AM

If you are reading those sizes based on dna gels and comparing with standards, don't read into it too much. I often ligate a 2kb insert into my expression vectors, and depending on whether the dna is methylated, a pcr product (not methylated), overloaded or underloaded, it can appear anywhere from ~1.5 to 2.0 kb. 

 

As bob suggested, no need to fuss around any more than you have and just sequence it from both ends. This will answer any and all ambiguities.


Edited by labtastic, 14 March 2015 - 11:49 AM.





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