Skip to content

Gibson Assembly Master Mix

March 1, 2013

It’s not the most exciting post but it may be useful to some. I’ve spent most of my time making plasmids and assembling TALEs since I’ve arrived in Australia. Most of the plasmids I’ve made with Gibson Assembly, which I must say is totally awesome. For sticking a single gene/ORF into an expression vector with two restriction sites, old-school cloning is still the way to go since you don’t have to PCR the entire plasmid.  But for anything more complicated, Gibson Assembly makes it a lot easier. I would suggest sequencing all the important parts of the plasmid after making a plasmid with Gibson Assembly.  Some weird stuff and PCR induced mutations can occur.

As far as the Master Mix, you can buy it from NEB or you can save money and buy all the components separately and save some money. It didn’t really make sense to me the way others were doing it so I recalculated everything and am doing it as described here:  Gibson Assembly Master Mix.  It worked better then the NEB master mix, but I have no idea why.

As far as primer design, this is how it goes for me:

1) I assembly the vector in silico using everyVector, which is a totally awesome tool (lots of totally awesome stuff in a boring post).

2) I make about a 44-mer oligo that span the fragment junctions and try to start it and end it in a G or C.  If there are a lot of A/Ts I make them longer, a lot of G/Cs I make them shorter.

3) Take the reverse-compliment of the oligo from step 2.

4) Trim about 12 bp off the 5′ ends of the oligos from step 2 and 3 so that the oligos are about 32 bp long.  This way I end up with 22 bp of complimentary sequence for the PCR and 20 bp of overlap between fragment for the Gibson Assembly reaction.

Not very scientific but it’s quick and works.

Advertisements

From → Posts

4 Comments
  1. Engin permalink

    Gibson Assembly does not PCR your plasmid. No. It does eat into your plasmid from the ends a little bit (and only in one strand), so you should sequence close to your insertion site (as you would with most sequencing primers). Definitely does not “PCR the entire plasmid”.

  2. ethanomics permalink

    Gibson Assembly does not PCR the entire plasmid but we usually end up PCRing the entire plasmid to make the fragments as we usually do not have restriction sites to linearize it in the way we want. That being said, one thing we are currently doing going forward is putting in unique restriction sites where we think might be useful in the future so we do not have to PCR entire plasmids. PCR induced mutations have be a annoying little issue we encounter from time to time, which has slowed us down. It is definitely something to think about and should be avoided if possible. Thanks for bringing that up.

  3. Alejandro permalink

    The link to the recipe for homemade Gibson assembly master mix self-references this post.

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s

%d bloggers like this: