quick change mutagenesis...... - (Apr/20/2013 )
I could not succeed in doing mutation....I redesigned the primers and tried from the beginning but i could not get...Im getting colonies after transformation but it is not having plasmid...Please help me regarding this...I have attached the pictures of my results n the protocol i used....In transformation im using controls too... kindly go through n give ur suggestions...
i think your PCR is working fine and i dnt think your Dpn digestion doing any harm here...you saying you didnt get plasmid, is it your procedure or the kit nor working?
Make sure your competent cells are good, in terms of sterility and efficiency?
besides this, what i would suggest is after Dpn digestion run the whole product, do a gel extraction, elute in 10 microlitre sterile water and transform. i usually transform half of the eluted volume or sometimes even the whole depending on the PCR yield and the efficiency of my competent cells.
Note: when u run the whole product after dpn digestion, sometimes u ll see lik a smear around the actual product, dnt panic lookin at that, jus cut around and do a gel extraction.
Thanks Gnana for your helping hand...I screened colonies doing plasmid isolation manually but im getting plasmid from the positive plate. Also im sure my competent cells are good since im getting good results in positive control. As per your advice i will try doing gel extraction and get back to you. Thanks....
Gnana...As per ur suggestion i tried gel extraction and transformed but again i got only one colony similar to the previous...all my transformation controls work...Can u plz help me to sort the problem...Where might be wrong?
how much plasmid you used for ctrl transformation and how many colonies you got in that? your competent cells commercial or lab made?
I transformed 1.5ug concentration of plasmid in control and i have got more than 1000 colonies from it. The competent cells im using are lab made by Cacl2 Mgcl2 protocol
Also after Dpn1 digestion i did not get any smear around the product as you said...I really dont know where will be the problem? plz do help me in sorting this Gnana...
Do you mean 1500 ng (1.5 microgram) of ctrl plasmid and got over 1000 colonies? if this is the case your cells are way down the required efficiency, which should be over 10^9 cfu/microgram plasmid. For mutageneis transformation i would say your cells shd atleast give 10^7 cfu per microgram. so, you got to prepare good cells and then proceed instead of repeating in the cells you already have.
About Dpn smear just dnt mind, even i dnt always visualize, here we cannot blame the digestion; if the problem is with inefficiecnt Dpn, you would still get colonies but wit wild-type plasmid.
if you want to ensure your competent cell efficiency, tranform some 10 pg of plasmid and count the colonies.
Is there any nice protocol to prepare this efficient competent cells other than Rbcl...Since in our lab it is not available....Can u give me a nice protocol?
you can find enough information to make good competent cells here http://openwetware.org/wiki/TOP10_chemically_competent_cells