producing recombinant -one gene plus strand/other gene complemenary and reverse - (Oct/30/2010 )
I have two fragments of my gene in different plasmids.I want to bring them together.But the second fragment in on complementary strand and in reverse orientation.How should i proceed?
It is very important that the two fragments are joined in frame. You do not want half of your protein to be scrambled. You can check that the two segments are in frame by hand or using a sequence management program (eg DNAstar, VectorNTI) or even a internet based DNA to Amino acid translation webtool.
As for how to join the two fragments you have a few choices
1. Restriction site ligation.
By chance the ends of both DNA fragments have restriction sites which allow the two to be ligated inframe. If you have that consider yourself lucky. The Goddess of Molecular biology looks fondly upon you. Offer a kit kat as a sacrifice so that your subsequent ligation will proceed without trouble.
Failing that you can use PCR to introduce that restriction site taking care to avoid changing the coded amino acid. Big primer can be purchased (~100bp). This mean if you can engineer upto 10 AA at the ends of your gene segments to produce the restriction site that can link the two protein coding sequences.
2.Ligation by PCR
You can amplify the DNA segments with primers that have homology to the next gene segment. Fragment 1 3' end has homology to the 5' end of Fragment2. Mix the two amplified segments in a PCR mix. Once melted the 3' end of Fragment 1 anneals to 5'end of Fragment 2. Then the DNA polymerase extends the the ends of each DNA fragment. (Imagine PCR with really big 5' overhangs).
3. Ligation by homologous recombination
Again you amplify the DNA using primers that contain sequence homology to the DNA segment adjacent to it.
Next add all the DNA fragment (vector and inserts) into
A - a yeast cell (by transformation). The yeast cell will use its homologous recombination machinery to build the plasmid. Extract plasmid DNA and transform back into bacteria. Your vector must be a shuttle vector, being able to grow in both yeast and bacteria.
B- a vial and use an vitro homologous recombination kit. Clontech sells one. The enzyme recombine the DNA fragment (vector and insert) into a plasmid. Transform plasmid into bacteria.
4. Ligation by (a lot more) money.
You lab is financially well to do. And you have determine that your time would be better spent doing real experiments. Go online and find a gene manufacturing company (eg Genscript, DNA 2.0, etc). They will build your synthetic gene, even optimising its sequence for gene expression. Submit your proteins AA sequence. Lab coughs up the cash and a week or so latter you have your gene. (The prices has dropped alot in recent years).
5. Ligation by scientific collaboration.
You have determine that your time would be better spent doing real experiments. And importantly you actually speak to people outside your lab. Specially you know somebody in a plasmid construction lab.
Go up to said person, "Hey <insert name>, I got a project that I have in mind <insert a few details>. It requires a person of your skills <optional:smile>. I am sure you find this very interesting and would like to collaborate. <Jedi mind trick / force lightning>" Note the use of force lightning is available only to those who have obtained the level of PI and can only be used on individuals 10 levels lower than yourself.
perneseblue on Sat Nov 6 16:13:26 2010 said:
It is very important that the two fragments are joined in frame. You do not want half of your protein to be scrambled. You can check that the two segments are in frame by hand or using a sequence management program (eg DNAstar, VectorNTI) or even a internet based DNA to Amino acid translation webtool.
As for how to join the two fragments you have a few choices
1. Restriction site ligation.
By chance the ends of both DNA fragments have restriction sites which allow the two to be ligated inframe. If you have that consider yourself lucky. The Goddess of Molecular biology looks fondly upon you. Offer a kit kat as a sacrifice so that your subsequent ligation will proceed without trouble.
Failing that you can use PCR to introduce that restriction site taking care to avoid changing the coded amino acid. Big primer can be purchased (~100bp). This mean if you can engineer upto 10 AA at the ends of your gene segments to produce the restriction site that can link the two protein coding sequences.
2.Ligation by PCR
You can amplify the DNA segments with primers that have homology to the next gene segment. Fragment 1 3' end has homology to the 5' end of Fragment2. Mix the two amplified segments in a PCR mix. Once melted the 3' end of Fragment 1 anneals to 5'end of Fragment 2. Then the DNA polymerase extends the the ends of each DNA fragment. (Imagine PCR with really big 5' overhangs).
3. Ligation by homologous recombination
Again you amplify the DNA using primers that contain sequence homology to the DNA segment adjacent to it.
Next add all the DNA fragment (vector and inserts) into
A - a yeast cell (by transformation). The yeast cell will use its homologous recombination machinery to build the plasmid. Extract plasmid DNA and transform back into bacteria. Your vector must be a shuttle vector, being able to grow in both yeast and bacteria.
B- a vial and use an vitro homologous recombination kit. Clontech sells one. The enzyme recombine the DNA fragment (vector and insert) into a plasmid. Transform plasmid into bacteria.
4. Ligation by (a lot more) money.
You lab is financially well to do. And you have determine that your time would be better spent doing real experiments. Go online and find a gene manufacturing company (eg Genscript, DNA 2.0, etc). They will build your synthetic gene, even optimising its sequence for gene expression. Submit your proteins AA sequence. Lab coughs up the cash and a week or so latter you have your gene. (The prices has dropped alot in recent years).
5. Ligation by scientific collaboration.
You have determine that your time would be better spent doing real experiments. And importantly you actually speak to people outside your lab. Specially you know somebody in a plasmid construction lab.
Go up to said person, "Hey <insert name>, I got a project that I have in mind <insert a few details>. It requires a person of your skills <optional:smile>. I am sure you find this very interesting and would like to collaborate. <Jedi mind trick / force lightning>" Note the use of force lightning is available only to those who have obtained the level of PI and can only be used on individuals 10 levels lower than yourself.
Hear hear!
perneseblue on Sat Nov 6 16:13:26 2010 said:
Go up to said person, "Hey <insert name>, I got a project that I have in mind <insert a few details>. It requires a person of your skills <optional:smile>. I am sure you find this very interesting and would like to collaborate. <Jedi mind trick / force lightning>" Note the use of force lightning is available only to those who have obtained the level of PI and can only be used on individuals 10 levels lower than yourself.
No wonder this trick was never worked for me because I used on individuals at least 20x levels upper than myself, because my PI don't want to use it...LOLx