gel purification or ligation problem
Posted 21 February 2009 - 05:27 PM
Posted 21 February 2009 - 08:54 PM
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.
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
Posted 21 February 2009 - 11:26 PM
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.
Posted 21 February 2009 - 11:46 PM
You may observe DNA band shift if ligation is successful.