My clone ( cDNA Clone) has 2 bp (C&A) deletion as a result their is frame shift and no protein expression is taking place. gDNA clone reveals no deletion but all my cDNA clone reveals 2bp deletion (C & A) at a particular site. Will it be feasible to use SDM for filling those bases? if not what other techniues can I use?
Site-directed mutagenesis could be an option, but it won't guarantee that no mutation will occur elswhere. I would rather start the cloning again, if feasible (i.e. if you still have enough template)
Where does the mutation occur ? If it lies at the border between vector and insert, it may be due to a faulty oligonucleotide primer. If it lies in the middle of your gene, then it may be caused by the poor fidelity of reverse transcriptase (or DNA polymerase, but this is less likely if you use a high fidelity polymerase, e.g. Phusion).
Another possiblity would be to order a synthetic gene, which will cost a few hundred $, but may still be a good value if you take into account the cost of reagents and time.
Site-directed mutagenesis could be an option, but it won't guarantee that no mutation will occur elswhere. I would rather start the cloning again, if feasible (i.e. if you still have enough template)
Where does the mutation occur ? If it lies at the border between vector and insert, it may be due to a faulty oligonucleotide primer. If it lies in the middle of your gene, then it may be caused by the poor fidelity of reverse transcriptase (or DNA polymerase, but this is less likely if you use a high fidelity polymerase, e.g. Phusion).
Another possiblity would be to order a synthetic gene, which will cost a few hundred $, but may still be a good value if you take into account the cost of reagents and time.
If you still have your original transformation plates, I'd just screen more colonies to find one that doesn't have the deletion. It's just as much work (if not more), to use site-directed mutagenesis as it would be to try the original cloning again. You might have to backtrack to making more cDNA first since it sounds like you have the same deletion in more than one colony.
Check your clone first before spending money on repairing what might be a tip of an iceberg. It looks that you are sequencing only the ends to verify you have the correct clone. Select more clones for checking the ends and use other means to verify your construct (PCR, RE) first.
The 2bp deletion maybe simply repaired by subcloning into a different vector using HPLC-purified primers. From what I see, you are selecting clones in the initial stage which is perfectly suitable for subcloning.
Pierre Béguin the mutation is in the middle of the gene.. and i have used Phusion polymerase for my amplication.. I think cloning again would be a better option..
Thanks
Katie A Burnette yes I have Original transformed plate in which very few colonies(24-25) appeared after transformation and out of those 25 colonies only 12 were positive, and i have checked them all.. all of them showing deletion of those particular base pairs..
It would be better to do the cloning again..
Thanks for ur suggestion.
Try Q5. I have sequenced many clones and the polymerase NEVER gave me a problem. With Phusion, I get problems all the time despite Phusion having next-to-the-best reputation.
As suggested by Kathy, check what you are assembling. If it is a fusion of separate parts, think about the strategy and if you want the fusion at all.
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