Talk:'Round-the-horn site-directed mutagenesis

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(New page: I would not recommend using desalted primers for this procedure, even if they are relatively short (~20 bp each)- I did so and got 1, 2, and 3 base pair deletions at the ligation site. I s...)
Current revision (11:20, 17 May 2010) (view source)
 
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I would not recommend using desalted primers for this procedure, even if they are relatively short (~20 bp each)- I did so and got 1, 2, and 3 base pair deletions at the ligation site. I sequenced 10 constructs and they were all like this, presumably due to truncated primers. Maybe add a warning to use purified primers? This needs to be factored into the tradeoffs for this method. Quikchange works quite well with deslated primers in my hands, probably because the mutation is in the middle of the primer. --[[User:Jonathan Lake|Jonathan Lake]] 12:19, 17 May 2010 (EDT)
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I would not recommend using desalted primers for this procedure, even if they are relatively short (~20 bp each). I did so and got 1, 2, and 3 base pair deletions at the ligation site. I sequenced 10 constructs and they were all like this, presumably due to truncated primers. Maybe add a warning to use purified primers? This needs to be factored into the tradeoffs for this method. Quikchange works quite well with desalted primers in my hands, probably because the mutation is in the middle of the primer. --[[User:Jonathan Lake|Jonathan Lake]] 12:19, 17 May 2010 (EDT)

Current revision

I would not recommend using desalted primers for this procedure, even if they are relatively short (~20 bp each). I did so and got 1, 2, and 3 base pair deletions at the ligation site. I sequenced 10 constructs and they were all like this, presumably due to truncated primers. Maybe add a warning to use purified primers? This needs to be factored into the tradeoffs for this method. Quikchange works quite well with desalted primers in my hands, probably because the mutation is in the middle of the primer. --Jonathan Lake 12:19, 17 May 2010 (EDT)

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