Talk:CH391L/S12/PCR and advanced PCR techniques
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- Brian Renda 15:47, 6 February 2012 (EST): Another cool PCR variant is called RPA (recombinase polymerase amplification), it is isothermal, finished in about 10-15 minutes, and the ingredients can be dried and stored at room temperature .
- Yi Kou 18:24, 6 February 2012 (EST):Really nice technique!
- Razan Alnahhas 23:12, 9 February 2012 (EST): Has PCR mediated DNA shuffling been successfully used in directed evolution, and are there only certain sequences that this technique can work on?
- Ben Slater 12:28, 10 February 2012 (EST): Hi Yi, I noticed you mentioned a few things about Phusion being both more accurate and faster than Taq. So what is the downside? Price? Does it have any different thermal sensitivity?
- Yi Kou 17:29, 11 February 2012 (EST): Phusion DNA Polymerase is a combination of the new pyroccocus like enzyme and a processivity domain, which tends to work better at higher denaturation and annealing temperatures due to higher salt contained in its buffer. Thermal sensitivity is a little different from the tradtional ones, as can be seen from here NEB phusion manual. For the downside, price is one factor, but I dont see any others. Personally, my experience tells me that this enzyme is pretty accurate and stable. It has been said that it could even stand on bench under room temperature for 2 months without losing much activity.
- David M. Truong 20:59, 12 February 2012 (EST): From personal experience, I've found that Phusion has a tendency to generate more non-specific products than simple Taq. However, the higher product yield generally overrides this concern if one were to just minimize the number of cycles used.
- Yi Kou 01:43, 13 February 2012 (EST): Thanks for sharing!
- Jeffrey E. Barrick 11:45, 11 February 2012 (EST): A lot of your links to Wikipedia have a formatting problem and are not working. Ex: "Khorana", "Overlap extension PCR"? Just get rid of the last / in the link and they will work.
- Yi Kou 17:29, 11 February 2012 (EST): Sorry for this! I have corrected them.
- Jeffrey E. Barrick 11:55, 11 February 2012 (EST):It's probably a good idea to link to Stratagene when talking about QuikChange.
- Yi Kou 17:29, 11 February 2012 (EST): Good idea it is. I have added a link.
- Jeffrey E. Barrick 11:55, 11 February 2012 (EST):Reverse PCR is actually a pretty interesting and useful technique for finding unknown sequences in a genome. I think you need to explain better that the restriction sites are not added by the experimenter (If they were, the sequence would already be known). Rather, one cuts at random throughout a genome with a RE with a common site, so that you generate < 5kb segments on average that PCR of the circularized construct can span. A similar approach is used in next-gen sequencing to make mate-paired libraries.
- Yi Kou 17:29, 11 February 2012 (EST): Yes, I added the explanation for that. I was also pretty confused at the time before your explanantion :)
- Piepenburg O, Williams CH, Stemple DL, and Armes NA. DNA detection using recombination proteins. PLoS Biol. 2006 Jul;4(7):e204. DOI:10.1371/journal.pbio.0040204 |
- Stemmer WP. Rapid evolution of a protein in vitro by DNA shuffling. Nature. 1994 Aug 4;370(6488):389-91. DOI:10.1038/370389a0 |