Once more... cloning of a 13 kb fragment into pBR322 - (Mar/07/2006 )
Hi everybody.
Since some weeks I am really trying to clone a 13 kb fragment into pBR322.
I do a sequential digestion with the 13 kb fragment. Nhe I and Sal I. After digestion, I checked the results and the purify the 13 kb fragment with the QiaEx II Kit. The problem is, that the recovery is not that much and i used to ligate it many times, with different rations ( Insert : Vector --> 3:1 ; 6:1, 8:1 , 1:3 , 1:1) and i used to try different cells like TOP10 F' , Stbl4, DH5alpha in transformation.
I doubt, that ligation works because there is no visible band at the size of the expected vector.
I also electroporated the cells but with a negative result. Does anyone has some good tips for further ligation and transformation? Thanks a lot in advance for any given suggestion.
some where along the line you're losing you 13kb fragment. You might consider increasing the amount of DNA in your digest or trouble shooting your gel extraction. If you digest a larger amount of dna it should increase your recovery accordingly.
Hi there,
please read this post in reagrds to your DNA. The longer your DNA the more likely DNA damage is if you use EtBr.
http://www.protocol-online.org/forums/inde...showtopic=14056
LeserattePD
Since some weeks I am really trying to clone a 13 kb fragment into pBR322.
I do a sequential digestion with the 13 kb fragment. Nhe I and Sal I. After digestion, I checked the results and the purify the 13 kb fragment with the QiaEx II Kit. The problem is, that the recovery is not that much and i used to ligate it many times, with different rations ( Insert : Vector --> 3:1 ; 6:1, 8:1 , 1:3 , 1:1) and i used to try different cells like TOP10 F' , Stbl4, DH5alpha in transformation.
I doubt, that ligation works because there is no visible band at the size of the expected vector.
I also electroporated the cells but with a negative result. Does anyone has some good tips for further ligation and transformation? Thanks a lot in advance for any given suggestion.