In some articles I have seen that for screening of a specific gene the authors just used a primer set and then sequenced the fragment. But, in other articles, after PCR, fragments were cloned and then sequenced. What is the difference here?
Please specify your question: Are you looking for mutations or what is the purpose of you PCR-based screening?
@ Sezar: Thanks
@Sabine: finding some gene responsible for the special function.
Your question is still not clear. Are you intending to look for the expression levels of a specific gene, mutations, ...? Just doing a PCR won't tell you anything about the special function of a specific gene. If you are seeking advice you have to provide some more details or simply ask you supervisor.
@Sabine Strehl: I think he is just confused by the preparation of sample for sequencing done in different papers . he want to ask the difference between sequencing of PCR purified template and The PCR template which is cloned in bacteria like DH5-Alpha.
@Mahdi- Sezer Okay has already given reason.
i want to add something in Sezer's answer that it is always preferred to Firstly clone your PCR product in Bacteria by inserting it in suitable Vector like p-Jet Cloning vector before Sequencing as:
1.) It reduces the chance of degradation of your PCR Product during transportation for sequencing as bacteria grow in 37 degree .
2) and when the PCR product is rare (i.e. we are not getting the same product again and again) we go for in-vivo cloning . as the cloning vector contains universal primers.
Hope this will help you.
All the best
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