Molecular cloning

[Zwiedagon]

I'm having trouble to clone some genes on DH5alpha E. coli strain. When I sequence the insert, I can only get 600 bp instead of the original size of the insert (1,2 kb). Does anyone know if this strain has restriction enzymes that can cut the insert?

[CharonY]

Did you sequence the inset or just determined size? The strain is restriction deficient. Most likely the ligation did not work as expected, or you had a mix in fragments and the smaller gets preferentially cloned etc. etc.

[BurningKrome]

I'm assuming you're discussing a PCR reaction to clone...and not discussing the process of sequencing the clone.

 

You may want to verify your melting and annealing temps using a calculator such as this one...

 

http://www.basic.northwestern.edu/biotools/oligocalc.html

 

It may be as simple as lengthening your extention time after annealing.

[dttom]

That means you got the insert but you cannot sequence it because it gave you just a 600bp piece? Then you may use another primer to continue the work, better to have some regions in overlap with the first 600bp, then you will get your first 600bp, and a second piece of another 600bp with some overlap so as to enable arrangement of fragments.