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gel purification or ligation problem - (Feb/21/2009 )

I have a 7.5kb vector that is cut with BamHI and XhoI to pop out an old 1kb insert and is to be used as a vector (now 6.5kb) for a new 1.3kb insert. The new insert is cut out of another vector using the same RE, BamhI and XhoI. Both digests give me two clear bands. I cut them out with clean razors and gel purify by Qiagen gel extraction kits. Both allow recovery of with decent A260/280 ~1.8-ish and concentrations of 15 to 40 ng/ul. I usually cut 2 ug of each plasmid to recover fragments for ligation. Then I recombine the fragments in 3:1, 5:1 insert: vector and run quick liagation (50ng vector and 30 to 50ng insert) with NEB buffer and enzyme for 20 min at RT. I transform 4ul into TOP10 cells for 30 min and heat shock 42C for 30 sec. add 250ul SOC and shake 1hr at 37C. I plate 65ul of transformants on pre-warmed LB amp plates and get no growth in either vector alone, vector plus insert OR vector no ligase. Both RE work completely since there is no linearized band, only the two digested products. I it seems as though ligation is the problem, but have noticed that my A260/230 is really low with the gel preps (compared to a Qiagen plasmid prep).
Any suggestions?

-yoda1311-

yoda1311 on Feb 21 2009, 05:27 PM said:

I have a 7.5kb vector that is cut with BamHI and XhoI to pop out an old 1kb insert and is to be used as a vector (now 6.5kb) for a new 1.3kb insert. The new insert is cut out of another vector using the same RE, BamhI and XhoI. Both digests give me two clear bands. I cut them out with clean razors and gel purify by Qiagen gel extraction kits. Both allow recovery of with decent A260/280 ~1.8-ish and concentrations of 15 to 40 ng/ul. I usually cut 2 ug of each plasmid to recover fragments for ligation. Then I recombine the fragments in 3:1, 5:1 insert: vector and run quick liagation (50ng vector and 30 to 50ng insert) with NEB buffer and enzyme for 20 min at RT. I transform 4ul into TOP10 cells for 30 min and heat shock 42C for 30 sec. add 250ul SOC and shake 1hr at 37C. I plate 65ul of transformants on pre-warmed LB amp plates and get no growth in either vector alone, vector plus insert OR vector no ligase. Both RE work completely since there is no linearized band, only the two digested products. I it seems as though ligation is the problem, but have noticed that my A260/230 is really low with the gel preps (compared to a Qiagen plasmid prep).
Any suggestions?


Ok yoda,

2 things. but first congrats on ur successful gel extraction and restriction digest.

1. vector only not working -> cell competency issue?!

2. low a260/230 is quite an issue from time to time for sequencing and ligation.


SuggestionL

for 1. u might want to make new batch of competent cell . coz the vector alone is a positive control to see if ur transformation is working or not.


2. for this , from my experience an EtoH wash works like a charm in improving the A260/A230

-hanming86-

Inaddition to other suggestions,

I would suggest to run the vector and insert on gel to get a better estimate of the concentration.

Also i would calculate the amount of insert in my ligation based on the insert size giving me 1:3 molar ratio of vector:insert.

Check if ligase and buffer are fine and working.

-scolix-

Hi,
You may observe DNA band shift if ligation is successful.

-WHR-