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Cloning-wrong expected insert size


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#1 thaonguyen

thaonguyen

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Posted 20 December 2013 - 08:53 AM

Dear all,

I tried so many months to get two plasmids by double recombinaison-using In-phusion 5x method that i were used to. It works really well.

Here is the strategy :

peGFPN1 is cut with Age1/HindIII.

two insert (100pb and 2kb) are generated by PCR. PCR products are run on agarose gel. Desired fragments are extracted.

Strategy.jpg

 

I got from 2 to 20 clones (varies for different mutants or different tries).

 

I redigested my clones with Age1/HindIII.

Normally, i should get two fragments : 4.7kb (vector) and 2.1kp(insert).

 

Actually, i got two fragments : 4.7kb (vector, i suppose) and 3kb (???). Multiple insertion seems not to be the case here.

 

Do you have some explanations and sugestions to solve my problem, please. I'm losing (lost) my faith.

 

Dearly thankful to all.

 

 






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