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This image shows how OE-PCR might be utilized to splice two DNA sequences (red and blue). The arrows represent the 3' ends. As in most PCR reactions, two primers—one for each end—are used per sequence. To splice two DNA molecules, special primers are used at the ends that are to be joined.
Two-step RT-PCR, as the name implies, occurs in two steps. First the reverse transcription and then the PCR. This method is more sensitive than the one-step method. Kits are also useful for two-step RT-PCR. Just as for one-step PCR, use only intact, high-quality RNA for the best results. The primer for two-step PCR does not have to be sequence ...
RT-PCR (or Reverse Transcription PCR) is used to reverse-transcribe and amplify RNA to cDNA. PCR is preceded by a reaction using reverse transcriptase, an enzyme that converts RNA into cDNA. The two reactions may be combined in a tube, with the initial heating step of PCR being used to inactivate the transcriptase. [4]
The cleavage of the tags with Xho1 and mixture of the different samples, followed by ligation, form the first concatenation step. The second step uses one of the restriction endonucleases with consensus to the adapter molecule attached to the 3’ end. They are again ligated, and PCR is performed to purify samples for the next joining. The ...
This method yields high-quality, largely double-stranded DNA which can be used for both PCR and RFLP analysis. This procedure can be automated [9] and has a high throughput, although lower than the phenol-chloroform method. This is a one-step method i.e. the entire procedure is completed in one tube.
RC-PCR provides significant advantages over other methods of amplicon library preparation methods. Most significantly it is a single closed tube reaction, this eliminates cross contamination associated with other two-step PCR approaches as well as utilising less reagent and requiring less labour to perform.
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