Actual size of parent plasmid where i released insert is 8.1 ie (5.8 backbone and 2.3insert size) and actual Plasmid size which i used as backbone was 8 (5kb is backbone and 3.0 is insert).
I had cloned a 2.3kb insert into 5.0kb backbone. It is directional cloning (just cut and paste) cohesive end cloning. For me 3:1 (insert:vector) ratio worked, mean got lot of colonies on Amp resistance plate. I did restriction digestion i was able to release insert and backbone separately. But the insert size hs become 2.3 to 2.4kb and backbone from 5 to 6kb. How this can happen.
I did colony PCR all my colonies are positive. I did Western Blot Protein is expressed. Then to sequence verify the clone i sequenced with vector specific primer and insert specific primers. Primers designed for Insert has given good read but backbone specific primers are not the giving read itself. And the Backbone specifc primer is BGH-REV. Please do help me i am clue less
Hi Bindhya, interesting problem. I guess that the vector is your problem. Otherwise it makes no sense that your vector primer gives no sequence. I recommend to get a new vector batch and clone your PCR fragment again. if you want to find out what's wrong, get sequencing primer directed out of your insert to get information about your vector
Hi Bindhya, interesting problem. I guess that the vector is your problem. Otherwise it makes no sense that your vector primer gives no sequence. I recommend to get a new vector batch and clone your PCR fragment again. if you want to find out what's wrong, get sequencing primer directed out of your insert to get information about your vector
Hi Bindhya,
Uwe's suggestion of sequencing out from the insert into the vector should tell you what's going on.
I can't be certain as there is some information missing from your cloning description, but I'd guess that it will show you that you have the parent plasmid, not the final construct you want. The sizes fit, at least for your digest. Are both plasmids Amp resistant?
-Richard
Hi Bindhiya,
Rearrangement s do occur in the plasmids during the process of cloning and excision of certain region is very commonly observed. You did colony PCR and found positive clones....I guess this was done using gene specific primers. It is better to use either plasmid specific primers (flanking the MCS or the region where gene is cloned) or a combination of a plasmid and a gene specific primers. In fact, one should always do a restriction analysis to get an idea of the overall backbone of the plasmid..
bye
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