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Nimblegen exome, Prepairing the Hybridization Sample
Hello everyone!
Today I had a mistake in the lab during the hybridization step of "Nimblegen EZ Library SR" protocol.
In the last part of it, we added the probe pool before denaturing the libraries pool.
In the protocol it says to denature 10 minutes at 95 degrees. As far as the biotin is ligated by a covalent bound to those probes, what we did was denaturing only 3 minutes at 95 degrees and finger crossed not to loose the biotin.
I've been reading several protocols about exome capture and in all of them goes first denaturation, then adding the probes pool. So then it has to be a very good reason to do it in this order.
I have already writen to the customer service to know about it.
At least we have enough libraries pool and reactives to repeat it, but we rather try not to loose the first try.
What do you think about it?
Thank you!
Today I had a mistake in the lab during the hybridization step of "Nimblegen EZ Library SR" protocol.
In the last part of it, we added the probe pool before denaturing the libraries pool.
In the protocol it says to denature 10 minutes at 95 degrees. As far as the biotin is ligated by a covalent bound to those probes, what we did was denaturing only 3 minutes at 95 degrees and finger crossed not to loose the biotin.
I've been reading several protocols about exome capture and in all of them goes first denaturation, then adding the probes pool. So then it has to be a very good reason to do it in this order.
I have already writen to the customer service to know about it.
At least we have enough libraries pool and reactives to repeat it, but we rather try not to loose the first try.
What do you think about it?
Thank you!
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