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Hi,
Very new to this, so I may be using wrong nomenclature. Being trying for a week now to get answers from Illumia or the internet without luck so I figured I would try to ask here.
I have some mammalian cells.
I treat them with a TALEN that generates a small deletion or insert (an indel, we are only interested in ones smaller than e.g. 10 bp) at a specific site in the genome of some of the cells.
I would like to quantify how often this happens and what the sequence of the region is.
Currently we do this by extracting genomic DNA from the pool of cells and then running a PCR. We then TOPO clone the PCR and pick bacterial clones and sequencing the PCR in the TOPO vector from the bacteria on a standard sanger sequencer.
This works but it takes a lot of work to get even a 100 sequences.
We would like to have many many more sequences and do it simultaneously with genomic DNA from cells that have been treated with different TALENS.
We have a MiSeq which I imagine would be able to sequence the PCR products directly if they had proper adapters and index sequences ligated (or as part of the PCR primer).
My questions are:
1) Is this the "correct" way to do this?
2) What are the sequences I would need to add to my PCR primers (or ligate to the final PCR product) for it to work in a MiSeq
3) How many different PCRs would I need to put in the MiSeq simultaneously in order to avoid the problem with low sequence difference?
Best,
Lasse
Very new to this, so I may be using wrong nomenclature. Being trying for a week now to get answers from Illumia or the internet without luck so I figured I would try to ask here.
I have some mammalian cells.
I treat them with a TALEN that generates a small deletion or insert (an indel, we are only interested in ones smaller than e.g. 10 bp) at a specific site in the genome of some of the cells.
I would like to quantify how often this happens and what the sequence of the region is.
Currently we do this by extracting genomic DNA from the pool of cells and then running a PCR. We then TOPO clone the PCR and pick bacterial clones and sequencing the PCR in the TOPO vector from the bacteria on a standard sanger sequencer.
This works but it takes a lot of work to get even a 100 sequences.
We would like to have many many more sequences and do it simultaneously with genomic DNA from cells that have been treated with different TALENS.
We have a MiSeq which I imagine would be able to sequence the PCR products directly if they had proper adapters and index sequences ligated (or as part of the PCR primer).
My questions are:
1) Is this the "correct" way to do this?
2) What are the sequences I would need to add to my PCR primers (or ligate to the final PCR product) for it to work in a MiSeq
3) How many different PCRs would I need to put in the MiSeq simultaneously in order to avoid the problem with low sequence difference?
Best,
Lasse
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