TA cloning vector:insert ratio

Question

I am currently doing TA cloning using puc19 (2.5kb) vector and an insert (1.45kb). I have tried 1:3 molar ratio for ligation but it did not work. So, any idea what is the recommended ligation ratio for TA cloning?

Answer 1

I will tell you a great secret: one of the best teachers in your educational process is "technical support".  

they want to give you answers all the time (and often they can), and they are great as "supplemental mentors".  

learn what they say, and if their instructions are not helpful, then seek the advice of your PI (last resort).  

if i  were you, i would check again the calculations you have for your insert.  is it double stranded.  is it single standed?  how do you quantitate your insert concentration?  etc.etc.

please post back and let us know how things are going.

best karma,

dumbdumb

Answer 2

If I remember correctly, most protocols suggest determining the optimum ratio empirically, trying three ratios;  3:1, 1:1, and 1:3.

Also, try two different temperature conditions: overnight at room temp, and overnight at 14C (I use a PCR thermocycler for the 14C reaction).

Good luck!  Cloning/subcloning can be a huge pain in the butt.