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#1 Re: rf-cloning troubles » Any suggestions? » 2014-05-25 06:27:49

I used the protocol described in: Methods Mol Biol. 2014;1116:73-87 with minor changes.
Give me an Email and I'll send it to you so you'll be able to upload it to the website.

#2 Re: rf-cloning troubles » Any suggestions? » 2014-05-22 11:29:25

Steve,

The simultaneous RF cloning work fine. I'v inserted two fragments into pBABE-Puro on the first try (mCherry instead of puromycin and another gene at the MCS).

Gal

#3 Re: rf-cloning troubles » Any suggestions? » 2014-05-07 13:57:14

Take a look at that:

Application of the Restriction-Free (RF) Cloning
for Multicomponents Assembly
Yoav Peleg and Tamar Unger
(Methods Mol Biol. 2014;1116:73-87)
or at: http://www.ncbi.nlm.nih.gov/pubmed/24395358

#5 rf-cloning troubles » Any suggestions? » 2014-05-04 07:06:29

galcafri
Replies: 9

Hi Steve,
First, thats really a great site and it helps a lot in designing RF.
I'm trying to design primers for inserting 2 genes into pBABe-puro plasmid (one into the MCS and second instead of Puro) .
The trouble is that in both genes the first 21 nucleotides are simmilar.
Do you have any suggestions to overcome it?
Maybe to design a longer primer for the gene?
Thanks
Gal

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