I have designed the primers for PCR cloning, but i was not getting any colonies after the ligation. One of our colleagues said, that the restriction digestion might not be working as there is no linker sequence before/after the restriction site in the primer. What measures should I take to be successful in the cloning?
$\begingroup$
$\endgroup$
2
-
$\begingroup$ You can add the restriction recognition site directly next to your sequence, this is not a problem. However, a lot of restriction enzymes have a lower or no activity, if the restriction site is directly at the end of a DNA strand and require some additional bases here. $\endgroup$– Chris ♦Commented Aug 23, 2016 at 6:52
-
$\begingroup$ i was using bamhI and hindIII for digestion ... do i need to order a new primer set ... and is there any specific linker sequence for respective restriction enzymes? $\endgroup$– saikrishna s bCommented Aug 23, 2016 at 7:42
Add a comment
|
1 Answer
$\begingroup$
$\endgroup$
0
This is the website you need, and yes, most of the time it's better to have several extra bases at the start of your primer.
https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
