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#1 2014-08-04 17:11:59

Jan
Member
Registered: 2014-04-15
Posts: 7

Amount of mega primer/plasmids second PCR.

Dear all,

whats the amount of mega primers you use for the second PCR?
Also 10μM  ?

Plasmid template?

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#2 2014-08-05 11:00:18

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 112

Re: Amount of mega primer/plasmids second PCR.

Hey Jan,
The amount of megaprimer to use is provided in the output as 'insert', and is expressed as 'ng' instead of a μM concentration.
-Steve

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#3 2014-08-07 19:32:11

Jan
Member
Registered: 2014-04-15
Posts: 7

Re: Amount of mega primer/plasmids second PCR.

beako1980 wrote:

Hey Jan,
The amount of megaprimer to use is provided in the output as 'insert', and is expressed as 'ng' instead of a μM concentration.
-Steve

Aha!
Yes I was looking here: http://www.rf-cloning.org/QandA.php, but indeed if you use the website to make the primers (where you put the plasmids) it tells you have much you need! haha , silly me.

Another question, not so much related to the restriction free cloning itself: what kind of kit do you use to do the gel extraction. THe kit I am using (for a different project) has low yields of DNA, too low.
Any suggestion?

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#4 2014-08-09 01:40:56

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 112

Re: Amount of mega primer/plasmids second PCR.

Any of the main commercial kits should be okay, although I have often suffered from low recovery as well. I've used GeneCleanII mostly (it's a glass milk kit). Your best bet is to fiddle with the primary PCR a bit, and get better amplification if possible. Load everything!

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