Errors shouldn't be a problem as this is Phusion proofreading polymerase,,,
Cycle conditions according to https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530
| Initial Denaturation | 98°C | 30 seconds |
| 25-35 Cycles | 98°C | 10 seconds |
| Final Extension | 72°C | 8 minutes |
| Hold | 4-10°C |
|
On Monday, July 15, 2013 3:40:45 AM UTC+2, phillyj wrote:
On Sunday, July 14, 2013 5:59:51 PM, shreyans chordia wrote:--
> If u want to avoid the 9kb band, try to use less PCR template as possible.
>
I was going to say that too. Usually, you never see the plasmid
template band after pcr. What's your pcr enzyme and cycle conditions?
I'm suprised at the size. I would think it might have been better to
create multiple fragments and pcr combine them. Most likely will be
less prone to errors.
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