|Register||Search||Today's Posts||Mark Forums Read|
|Molecular Cloning Forum DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.|
| ||LinkBack||Thread Tools||Display Modes|
I've hit a snag, im new to my lab and have been assigned a project to investigate te effect of single amino acid changes in a tyrosine kinase. this is my plan of attack
1. extract RNA ffrom tissue and create cDNA
2. PCR amplify gene of interest using primers (do i need to include the start codon in my primer?)
3. insert into an expression vector - overproduction and purification
If you are expressing this protein in mammalian cells either your PCR product or the expression vector must contain a 5' Kozak consensus sequence and a start codon in addition to whatever promoter it uses (many mammalian expression vectors use a CMV promoter).
If you are expressing this protein in bacteria, you have to make sure your PCR products (i.e. your primer) includes a Shine-Dalgarno consensus sequence and a start codon.
Furthermore, you should make sure there is a stop codon in your PCR product or in the vector backbone so that you obtain the correct protein.
If you are expressing your protein as a fusion with another protein (like GFP, GSP, 6xHis, Myc, MBP, etc.), your coding sequence must be in frame with the tag, otherwise you will be making something other than what you expect. If the tag is C-terminal, then clearly you should not include a stop codon and if the tag is N-terminal, you should not have a Kozak sequence intervening between the tag and your first codon.
thanks for that, we're eventually going to do kinase assays, and we'll be expressing in E.coli. Does the shine-Dalgrano sequence need to be upstream of my RE1 site and start codon?
also, when inserting gene of interest into vector with his tag do i need to design new primers for expression that don't include the stop codon?
ugh I'm so confused - been out of the field for a while so its like starting from scratch!
If the His-tag is part of the vector, is it N'terminus or C'terminus?
If it is N' terminus then obviously the stop codon is ok. Furthermore, if the tag is N'-terminus, then there is a Shine Dalgarno and start ATG ahead of it, so you don't need to worry about adding them to your cDNA. However, you must design primers so that the gene is in frame with the tag. Otherwise you'll have an entirely different protein product than what you expect. The important primer here is the forward primer. The reverse primer doesn't need to make anything in frame with anything else because it's at the end.
For example, suppose my sequence starts out like this:
ATG GAG GGG CGG
And suppose in the vector, the sequence after the His tag has an MCS with EcoRI that looks like this:
CAT CAT CAT CAT CAT CAT GAA TTC
As you can see the frame of the restriction site is just GAA'TTC. So when you design a primer to add the restriction site, it must look like this:
However, suppose the vector was like this instead:
CAT CAT CAT CAT CAT CAT GGA ATT CGG
If I just clone in my sequence as is it will be out of frame:
CAT CAT CAT CAT CAT CAT GGA ATT CAT GGA GGG GCG G
In order to keep it in frame with the tag, I will have to make a primer that perhaps adds a nucleotide or two to maintain the frame:
GA ATT C__ ATG GAG GGG CGG
In this case, as you can see I need 2 nucleotides to maintain the frame. They could be the same ones from the vector backbone I suppose.
GA AAT CGG ATG GAG GGG CGG etc.
Now, when I perform the ligation, this will be in the appropriate reading frame. I should obviously sequence my final construct to make sure that the gene of interest is in frame with the His tag.
On the other hand, if the Hist tag C'terminus then the stop codon would interrupt your protein and the tag, so you'd need to remove it by designing new primers. Furthermore, you would need to make sure you have a Shine-Dalgarno and start ATG ahead of your gene. And finally, your reverse primer must maintain the reading frame as above with the C' terminal tag.
|Thread||Thread Starter||Forum||Replies||Last Post|
|Help needed on aminoallyl-labeled cDNA||palver||Molecular Biology Articles and Protocols||0||01-07-2009 07:40 AM|
|cDNA Synthesis and Amplification of POLY(A) mRNA by RT-PCR Protocol||domba||Real-Time PCR and Quantitative PCR Forum||3||09-09-2008 02:48 PM|
|Commercial cDNA or cdna Synthesized Random hemer for Transfection||bhunni||DNA Techniques||0||09-01-2007 07:12 AM|
|cDNA precipitation without coprecipitation of primers||Simone Marker||Protocols and Methods Forum||1||03-05-2007 07:36 PM|
|Optimal way to construct a YEAST 2-hybrid cDNA library?||Apuzzlepiece@gmail.com||Protocols and Methods Forum||0||08-10-2005 03:22 AM|