Dear Shoyab

how much dna template did you used for the pcr? because generally to avoid backgruond the template is very low (eg 0.1-1ng/pcr) and not detectable in agarose gel. From your results it seems that you use a lot of template and it is all digested by sono but your pcr do not work.

however i would like to suggest you to use PIPE cloning instead commercial kit for deletion, short insertion and mutagenesis.

You can find details about it on my blog:

https://proteocool.blogspot.com/?m=1

ProteoCool n1: Cloning methods overwiev

ProteoCool n3 : Primer design for PIPE cloning

good luck

Manuele

Dear Shoyab,

Strategene kit manual recommends to go for transformation after Dpn-1 digestion as the amplified product may or may not be visible on the gel (depending on the quantity of the amplified product)....personally, i would avoid transformation in such a scenario and will start a fresh experiment from the scratch with some optimisation (Check your annealing temperature as it depends on the sequence of your primers , extention time depends on the size of the construct). Regarding the visibility of the amplified product (after your mutagenesis reaction) it is most likely (with 95% surety) your template because after digestion with Dpn-1 nothing is visible ( Dpn-1 digests methylated and hemimethylated DNA/template). Don't run the mutagenesis product on gel (save some chemicals and time) until it is digested with Dpn-1, if you see a band after digestion go for transformation.

Dear All

Thanks for your reply.I did repeat the experiment with modified protocol.And i did get mutation.

@Shoyab Ansari what did you change to get the mutation to work?