Help designing primers for use in In-fushion cloning - (Oct/12/2012 )

I'm using the In-fushion HD cloning kit, and I'm a little confused on how to make the primers.
I understand I need to make the 15 base pairs of homology to the plasmid and our gene of interest, but what I'm really concerned about is how to keep things in frame, especially at the point where the plasmid meets our gene.

I've done one In-Fusion cloning ever, but I think I can help.

When designing your primers, you just need to keep in mind that the regions in your primers that are homologous to your vector will be recombined without any shifts of frame. You need to look for where your restriction enzyme is going to cut the vector, then shift the frame accordingly. I believe most cut after the first base of its recognition sequence. If that first base of recognition sequence is the last base of a codon, then you don't need to frameshift your insert sequence. Just paste it right in there in frame. If the first base of the recognition sequence is the first base of a codon, you need to add two bases to your insert to keep it in frame (and if it's the 2nd base of a codon, then you need to add one base).

I'll illustrate with an example:

Vector sequence around restriction site:

...GCCACC-ATG-ATC-CGT-CAT-GGATCCCGACATGTTACGTAG... (Kozak is underlined, bold is BamHI site for insertion, put dashes between codons for clarity of reading frame)

Cutting with BamHI will give:

...GCCACC-ATG-ATC-CGT-CAT-G and GATCCCGACATGTTACGTAG...

To combine your insert via InFusion cloning, you'll have 15 bases on either side of the cut site in your primers:

5'-CCATGATCCGTCATG+2 bases to put in frame+in frame homology to your insert sequence-3' (forward primer)
3'-CTACGTAACATGTCGGGATC+homology to end of insert sequence-3' (reverse primer) (frame doesn't matter here, but don't forget stop codon)

which will give you in your final product (vector+insert):

...GCCACC-ATG-ATC-CGT-CAT-GXX-(insert sequence in frame)-GATCCCGACATGTTACGTAG... (X's are the two extra bases you added to your primer)

Again (hopefully this isn't too redundant), to keep your insert in frame, you look at where the restriction enzyme will cut (for BamHI it's after the first base). If it cuts between two codons, then you don't need to frameshift anything. Just make a primer with your 15 bases of homology upstream of the cut and add your insert homology sequence in frame. If your restriction enzyme cuts after the first base of a codon in the vector (as the example above), then you need to add two bases before your insert sequence to keep it in frame (and you need to add one base if your enzyme cuts after the 2nd base of a codon).

I hope that helps (and isn't too late since you asked the question several days ago).

Just a quick follow-up with my earlier reply, after I've looked at my old InFusion cloning notes.

I ended up including the entire restriction site sequence in my primers on both ends. So if you wanted to design your primers that way, you would just adjust your insert sequence based on what frame your vector homology sequence is in (if your restriction site is in frame with your translated sequence, don't add any bases).

I hope that makes sense.

John Forsberg on Mon Oct 15 13:45:01 2012 said:

Hi thanks for the help! So to follow your example, if I cut with BamHI (which cuts after the first base of a codon) then I should fill in the the next 2 bases to keep the codon in frame, then use the start of the next codon for my gene of interest? Any advice on what I should fill in the next 2 base pairs in the BamHI codon?
I hope I'm making sense and thanks again.

Smog187 on Wed Oct 17 01:43:56 2012 said:

I believe I put the entire restriction sites into my primers because then I could easily cut out my insert at the end of cloning to confirm it went in (ended up with a BamHI site on either side of my insert, so I could just cut with BamHI to see my insert on a gel).

So if I were you, I'd put the entire restriction site on both of your primers, then add bases on the forward primer to keep it in frame. If your restriction site has codons split like this: ...G-GAT-CC... (with the dashes marking where the codons are), then you'll want to add one base after the restriction site to keep your insert sequence in frame. If the codons are like this: ...-GGA-TCC-... then it's in frame already, and you don't add anything before your insert sequence. If the codons are like this: ...GG-ATC-C... then you'll need to add two bases to put your insert in frame. But you definitely need to mark it out from the AUG in your vector to your restriction site.

Come to think of it, this would probably be easier if you pasted the sequences your using here.

As for what bases to use to fill in, I usually try to put in Alanine or Glycine residues whenever possible. If you're really lucky, the "leftover" residues from your restriction site will match the first codon in your insert (e.g. with a frame of ...G-GAT-CC... your first insert amino acid is Proline, so you can add any nucleotide to get a Proline out of the CC_ codon, then continue on with the rest of your insert sequence; or if your frame is ...GG-ATC-C..., and the next residue in your insert is Leucine, Histidine, Glutamine, Proline, or Arginine, you can add the two bases necessary then continue with your next residue).

All of this depends on what restriction site you're using, where it's located after your start codon (unless of course you're inserting into the MCS and your insert will have it's own Kozak and start codon) and what your insert sequence is after the restriction site. I will say it's preferable to avoid putting Proline residues into your sequence if you can avoid it (but if your frame really is G-GAT-CC, there's no avoiding it without destroying that BamHI site).

Make sense?

(oh, and sorry it took me so long to respond. I hadn't noticed you replied until tonight)