Adding restriction enzyme sites to sequences - (Nov/29/2010 )

Hey guys,

Im using a PET28c vector with a GFP insert. I need to remove the GFP and add a Galactose Oxidase instead. Im restricting the vector with Nde1 and HindIII to remove the GFP and need to create the same restrcition sites on the coding sequence of Galactose Oxidase, inorder to ligate the vector and GP together.

First of all, is my approach right?

Also, How do I go about introducing Nde1 site on the 5' end and the HindIII site on the 3' end along with a stop codon? I was thinking I'd use this website:
http://depts.washington.edu/bakerpg/primertemp/primertemp.html to design the primers but Im not quite sure how to design a primer with the restriction enzyme site included. If any of you geniuses could help, it would be great!

Thanks
- Mon -

Basically your primer will look like this:

5'spacer(3-6bp)_REsite_spacer_kozaksequence_specific-sequence 3' (i.e. your primer against coding sequence). The RE sites etc. always go on the 5' end of the primer.

So your forward primer will be:

ctagtaCATAGTctagtaGCCACC

underlined is Nde1 site, uppercase is kozak sequence.

Don't forget to reverse complement the reverse primer, but not the RE site.

Thanks a ton!

One question though, do I not need a restriction enzyme site on the 3' end? Since my vector uses a hindIII site at its 3' end, I was thinking I'll attach a hindIII site before the end codon and design a primer. What do you think?

Yes, you still need a RE site on the 3' end of the coding sequence, but for the primer this will still be on the 5' end as the specific primer sequence is the reverse complement of the coding sequence...

forward primer: 5'-ATGATGATGATG-3'
coding sequence: 5'-ATGATGATGATG---------------------TAATAATAATAATAA-3'
Reverse primer: 3'-ATTATTATTATTATT-5'

If you put the RE site on the 3' end your PCR will not work, or will be very inefficient and not give you what you want.

I would add the RE site after the stop codon, otherwise you will change the coding sequence of the protein and, how will the the protein know where to stop expressing?

Okay so when I take the initial region and add my restriction enzyme site alonf with the start codon, this is what I get:

Tm = 79.8 degrees C, Bases = 51, GC content = 56.9%, mismatched bases = 6
Primer Sequence 5' to 3':CCGTAGGACTGGAATCCTGAGCCTTCGACATATGGCCTCAGCACCTATCGG

Complement 5' to 3':CCGATAGGTGCTGAGGCCATATGTCGAAGGCTCAGGATTCCAGTCCTACGG

When I take the last bit of the region and add my stop codon and RE site, this is what I get:

Tm = 77.2 degrees C, Bases = 49, GC content = 53.1%, mismatched bases = 6
Primer Sequence 5' to 3':GGTGTTCCTAGTGTGGCTCGACGATCGCGTCTCAGTGAAGCTTGATTTG

Complement 5' to 3': CAAATCAAGCTTCACTGAGACGCGATCGTCGAGCCACACTAGGAACACC


So do I just delete the bit that says Complement 5'-3'?

Thanks

Okay you are perfectly right!

I have modified my primer to include the stop codon before the RE site, so heres what they look like:

Forwd primer to cctgagccttcgaCATATGgcctcagcacc:
Tm = 83.6 degrees C, Bases = 30, GC content = 60.0%
Primer Sequence 5' to 3':CCTGAGCCTTCGACATATGGCCTCAGCACC

Complement 5' to 3': GGTGCTGAGGCCATATGTCGAAGGCTCAGG

Rev primer to the seq cgcgttactcagtgaTGAAAGCTTtttgttaggaagccaag:
Tm = 83.0 degrees C, Bases = 41, GC content = 43.9%
Primer Sequence 5' to 3':CGCGTTACTCAGTGATGAAAGCTTTTTGTTAGGAAGCCAAG

Complement 5' to 3': CTTGGCTTCCTAACAAAAAGCTTTCATCACTGAGTAACGCG

Should I remove the bit that says complement 5'-3'?

Also, if you can pleease answer one last question:

Why are we using a version of pET28c that already contains an insert? (GFP in this case) and why do we then excise the GFP out to include our sequence of interest? Is it because it is a confirmation that our protein will be expressed?

Thanks

If you could show which bits are the specific sequence and which bits are the restriction sites (and which RE they are) that would be helpful. If you can also post the 30-40 bases each of the 5' and 3' ends of your sequence, it will be much easier to check whether the primers are correct.

Re GFP:

Sometimes people make tagged proteins that can be detected by various means. GFP tagging is one method that allows detection of proteins by using fluorescence, usually used for determining protein localisation in the cell. I suspect they are getting you to remove the GFP because they don't want a tagged protein in this case and they don't have an "empty" vector. You can also use the GFP plasmid as a transfection control, to show that your transfections are working properly if you are having problems detecting your protein of interest after transfection.