Gibson assembly master mix

[flight505]

Hello, 

The lab i am working in only has peg 6000, the miller lab protocol for Gibson assembly master mix uses peg 8000 

can i adjust for the molecular weight deference and use peg 6000 instead ? 

does anyone have experience with this ? 

Best regards, 

[Cathal Garvey]

Once you're into long-chain PEG, the difference is often not such a big
deal. Try it with the same weight *or* molarity, ideally both, and see
if it works OK with either.

I'm sure there'll be guides on selecting chain-length for other
protocols, but I wouldn't be surprised if it's more of an art than a
science.

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[Sebastian Cocioba]

Also, IIRC, chain length is the average length. Maybe it will work but
with low efficiency?

I seldom if ever check molar ratio of backbone to insert for ligation
and never had one fail on me so maybe you can get lucky with small
substitutions? Like Cathal said, its most likely more of an art than a
strict science. On the other hand, at the current street price for
GibbyRxns I would rather play it safe. Do let us know if you attempt it
and if it works. Its nice to add hacks and mods to the list of
protocols.

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D From: Cathal Garvey
Sent: 2/17/2014 5:51 PM
To: diy...@googlegroups.com
Subject: Re: [DIYbio] Gibson assembly master mix

[Cathal Garvey]

"Street Price" makes Gibson reactions sound far cooler than they deserve!

24.2% of funding goal, with 24 days left:

[DIYBIO Groningen]

It works well also with PEG6000. You can have some problem only in case your DNA inserts are very long like >5Kb.

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[Melissa Romich]

Hello,

i have the same problem as you. Have you tested PEG-6000 yet? If you had tested, maybe you can send me some more information. Lenght of your Fragments you want so ligate and if you had tested the same fragments with PEG-8000.
Thanks in advance!

Melissa

[Melissa Romich]

Hey,

i had worked with PEG-6000 and only some ligation worked. I have ligated 155bp + 3000bp +5080bp and get a fragment. Another mix with 3000bp + 5809bp did not work. Why do you think, that i will get problems with Fragments longer than 5kb? Maybe you could send me some more information about your fragments and the results.

Thanks in advance!

Melissa