golden gate cloning - (Aug/30/2013 )

Hi;

I'm struggling to understand the Golden Gate scheme. I dont work with TAL effectors, I need to make a repetitive string of small fluorescent proteins.

 

If I understand correctly, you need the overhang part on each end of each of the pieces you want to connect? so how having those four letter overhangs doesnt mess up with my reading frame? and also, im getitng four random letter every repeat in my protein?

They also say the enzymes like BsaI leave no scar, but they have one base that needs to be constant, so how come there is no scar?

 

I would like to have something like this:

<-plasmid>

 

Could you, please, give me a for-dummies explanation on how to design primers?

I have the piece that is my actual gene to be repeated, 

I think, if I understand it correctly, I need to get several sets of PCR primers and copy that gene with primers introducing recognition sites for Bsal (or similar restriction enzyme) and overhangs to connect my pieces in a special manner. But then, those overhangs I use stay there. How can a 4 letter overhang be used and not mess up reading frames for repeated genes?

 

Thank you very much for all help!

 

this webpage explains primer design for pcr.

 

this webpage will help you design primers.

Thanks for the reply.

I'm sorry I wasnt clearer with my question.

I've done plenty of PCR, and once I have sequences, I can design the primers.

 

What I don't know is what sequences do I need, and that will be the output.

 

For example, here http://m.nar.oxfordjournals.org/content/39/12/e82/F2.large.jpg the step 1 shows overhangs, but in the resulting assembled plasmid showed right underneath it, the connections lok semales. Where did those overhangs go?

 

Thanks!

 

 

 

 

 

mdfenko on Fri Aug 30 20:57:32 2013 said:

this webpage explains primer design for pcr.

 

this webpage will help you design primers.

Not sure if you've read this

http://j5.jbei.org/j5manual/pages/23.html

 

BsaI is a type II endonuclease and cuts somewhere downstream of its recognition site. It allows you to customise your overhanging end sequence. What you need to do is design primers such that the overhanging sequence is part of your gene sequence so that you don't introduce extra bases into your gene. For the adjacent gene, do the same with the complementary sequence on the complementary strand. Even though there is a base before the cleavage site, the base will be part of the fragment that you will be discarding. After BsaI digestion, you will get two fragments with complementary ends that will join at the junction of the two genes.

 

What do you mean by repetitive string of proteins? Fusion proteins? If so, you need to remove the TAA from the end of the gene.

 

Say, gene 1 ends with ACGGCCTAA and gene 2 starts with AGTTTC, you should design primers to get fragments like this (add extra bases to the ends of the primers for more efficient digestion).

 

Fragment for gene 1        Fragment for gene 2

....ACGGCCAGTTNGAGACC      GGTCTCNAGTTTC.....     

....TGCCGGTCAANCTCTGG      CCAGAGNTCAAAG.....

 

Since BsaI cuts like this (after the N's):

GGTCTCNXXXXX

CCAGAGNNNNNX

 

The resulting fragments will be:

 

Fragment for gene 1              Fragment for gene 2

....ACGGCC    AGTTNGAGACC      GGTCTCN    AGTTTC.....     

....TGCCGGTCAA    NCTCTGG      CCAGAGNTCAA    AG.....

 

See how the fragments at the far left and far right now have complementary sticky ends and fragments with the recognition site are discarded?

 

Hope that helps.

 

 

EDIT: Oops, it's been almost one month. Guess you've already figured out. Hope that helps anyway :)