Adding restriction enzyme sites to sequences
Posted 29 November 2010 - 01:28 PM
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.washing...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!
- Mon -
Posted 29 November 2010 - 03:13 PM
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:
underlined is Nde1 site, uppercase is kozak sequence.
Don't forget to reverse complement the reverse primer, but not the RE site.
Posted 30 November 2010 - 12:16 PM
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?
Posted 30 November 2010 - 02:44 PM
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?
Edited by bob1, 30 November 2010 - 02:46 PM.
Posted 30 November 2010 - 03:51 PM
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'?
Posted 30 November 2010 - 04:37 PM
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'?
Posted 30 November 2010 - 04:47 PM
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?
Posted 01 December 2010 - 02:56 PM
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.