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Problems cloning Hairpin vector


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#1 Marlow

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Posted 21 March 2009 - 11:24 AM

Hello first at all,
my job is to clone a long hairpin vector for a RNAi approach.
I cloned a sense part and a linker fragment together. Also i cloned via TA cloning a antisense Fragment in another vector (same type).
After that i want to open the bigger vector (sense+linker) with two enzymes and ligate the antisense fragment which is cutted out with the same combination. I checked with colony pcr my plates, but i didn't get any bands, expected for two colonies where it seems that sense and linker are in the vector.
I'm not sure what happend during the ligation, because the colonies surely got plasmids, but with screening primers i didn't get any result (expected for the two conlonies).
I repeated the ligation which differents amounts of insert and plasmid and also included a negative control. I'm kinda stuck at the moment.
Do you have any ideas why i can't verify the existience of an insert but also got plasmids inside ? (The whole lab is working with the same plasmid, if it would be an contamination problem i should at least amplify something). The positive controll for the colony pcr is working fine.

Thanks in advantage for your help.

#2 scolix

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Posted 21 March 2009 - 12:44 PM

You might have to use DMSO to get the PCr working because of the hairpin sequence.

You could do a diagnostic digest and also later sequence it to confirm the insert

#3 Marlow

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Posted 21 March 2009 - 01:19 PM

Well this is a good advice, do you have any idea of the right DMSO concentration ? Anyway I will do the diagnostic digest tomorrow.

#4 scolix

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Posted 21 March 2009 - 10:26 PM

you have to try different conc. of DMSO to optimise the pcr.




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