problem with cloning of dsred plz help - vector cut seems fine, still religation oocurs (Jun/14/2010 )
adrian kohsf on Jun 18 2010, 01:25 PM said:
I am using right antibiotic and its not expired.
comp cells too r freshly made and tested for any contamination.
Its all fine.
1. Did your 80 colony background come from vector plus ligase (incomplete SAP) or vector minus ligase (uncut vector contamination).
2. Typically, unless you vector plus insert plate has more than twice as many colonies as your vector plus ligase, most (if not all) of the colonies you pick will be vector only.
3. I was a firm believer in gel-purification of vector and insert for many years. But then I discovered that using a Spin-X cartridge (with a rinse step afterwards) was sufficient to purify vectors that were double-digested in the multiple cloning site. The cloning site fragment goes through the Spin-X, leaving vector behind. This method does require that you have achieved 100% linearization of the plasmid.
You report 1:3 and 1:5 ratios (insert:vector or vector:insert). The size of the DNA fragments is important in this calculation. The routine 3:1 insert:vector assumes that you have a 1 kb insert into a 3 kb vector.
tfitzwater on Jun 23 2010, 12:16 AM said:
2. Typically, unless you vector plus insert plate has more than twice as many colonies as your vector plus ligase, most (if not all) of the colonies you pick will be vector only.
3. I was a firm believer in gel-purification of vector and insert for many years. But then I discovered that using a Spin-X cartridge (with a rinse step afterwards) was sufficient to purify vectors that were double-digested in the multiple cloning site. The cloning site fragment goes through the Spin-X, leaving vector behind. This method does require that you have achieved 100% linearization of the plasmid.
You report 1:3 and 1:5 ratios (insert:vector or vector:insert). The size of the DNA fragments is important in this calculation. The routine 3:1 insert:vector assumes that you have a 1 kb insert into a 3 kb vector.
80 colonies came from vector plus ligase. Yes ur right most of them are vector only now. Can u give me more information about Spin-X catridge?
I set up vector to insert ratio of 1:3 and 1:5. not other way around. I calculated molar ratio and then set up the ligation accordingly.
thaithanhthuy on Jun 28 2010, 01:12 AM said:
I can see more colonies on control plates than on transformed plates.
I picked some colonies to screen, but am not sure how it goes as there are colonies on controls too..
Please suggest me why is that am getting colonies on control plates even after SAP treatment?
@HomeBrew: i re did the way u suggested and used CIP instead of SAP, i got the clone, sequenced too..
Thank u so much.