PCR Primers for introducing tag - (Sep/22/2008 )

Hi, I have recently cloned my wildtype gene in a pUAST vector. I want to introduce HA tags right before the gene and right after the gene(as two separate constructs). I also want o make sure I have a Kozak sequence and an A at the -3 before the tag. How can I do this by PCR. Can anyone suggest?

-predoc-

Hi predoc!

It's really easy! The HA tag is about 27-30bp, isn't it? Just order longer oligos then.

Forward primer:

5'-(3random bp)-(Restriction enzyme site)-(HA tag)-(start of your gene (20-30bp))

Reverse primer:

3'-(end of gene)-(HAtag)-(RE site)-(3 random bp)-

After the PCR cut the product with restriction enzymes and paste in the place of your unmodified gene.

Usually the region, complementary to the gene is around 23-26bp. You can add your Kozak in front of the HA tag. Take care of the START and STOP codons if you want the tags to be translated.

I do it like this all the time. I have added tags as long as 60bp with primers. In such a case I just ordered a 85bp oligo (HPLC purified) and no problem. I usually use Pfu for such PCR.

-Ramses-

Thanks Ramses. Will the PCR work if my plasmid is ~10kb? IS there a good Polymerase that will not introduce random mutations?

Thanks

-predoc-

QUOTE (Ramses @ Sep 22 2008, 10:23 AM)

I too have used this method successfully and easily to introduce HA tags. Just one addition, you may need to do a ramp-up pcr in order to get efficient product. This is where you start with a low annealing and have it increase with each round. At first your primer will not anneal to the entire plasmid since the HA and restriction site are not complimentary but once you get a bit of product this becomes the new template which is complementary to the entire primer. Otherwise you can do a nested pcr where you take the product of the first pcr, which may not be all that much, and use it as the template in a second pcr. This should give you a much more robust reaction.

-rkay447-

Thanks

-predoc-

can try Phusion. Great enzyme. KOD long template is also another good enzyme.

If you are worried about the 10kb PCR reaction, look for a unique restriction site in your gene.
ie (Using NotI and BamHI as examples)

Plasmid----5'Gene ---- NotI-----Gene 3'---BamHI---Plasmid

Built a forward primer that binds to this unique restriction site within your gene.
And build a reverse primer as described by Ramses. (Although I would use 6bp as guard rather than 3bp)

PCR amplify and you will get
Guard---NotI------Gene3'-HA Tag-BamHI--Guard

Cut this PCR product with the appropriate restriction enzymes. Cut the plasmid with the same enzymes, remove the DNA fragment that contains the old 3' end of the gene. And then ligate them plasmid and PCR product together.

Plasmid----5'Gene ---- NotI-----Gene 3'-HA Tag---BamHI---Plasmid

-perneseblue-