A few questions about insertion into a plasmid - (Sep/13/2005 )
Hi. I'm new in all of these and I need some help from the experts.
I need to introduce a sequence (750bp) into a plasmid. Basicly, My plan is:
To obtain the sequence I will use primers upstream and downstream from the ends and hopefully I will get ~800bp.
To introduce this sequence into a plasmid vector, I incubate the plasmid with one of the restriction enzymes which don't cut my searched sequence. As both have complementary sticky ends they will join and I will get my sequence into the plasmid.
Finally, I need to express this plasmid into my cell line and I will get the protein within the cell.
Are all these steps OK?
Could anyone tell me what more things I should be aware of?
If I use RNA from different cells that those where I will express the sequence into (in any case all human cells), what problems can I have? My assumption is that the human DNA is the same regardless of the cell, so I will have no problems.
i would say yes if you voluntary didn't mention that you need to cut your PCR product by the appropriates enzymes.
After that point, the things you need to care is :
antibiotics for bacteria culture
antibiotic for cell culture
dam or dcm sensitive restriction sites.
Thank you for your reply. I didn't mention involuntary because I was confused about TA cloning or using restriction enzymes.
Do you think I should try with TA cloning? It seems easier to me. What problems can come up doing that?
About play with DNA from different human cell lines, is it a proper scientific method?
For T/A cloning you need to be sure your polymerase gives the A overhang. Taq polymerase and several blends of Taq and a proofreading enzyme do so, others don't.
Apart from that, when doing T/A cloning, which is very efficient, you need to check the direction of your insertion (by restriction analysis or a PCR with primers that anneal on your insert and on the T/A cloning vector).
Then, you need to be sure that your insertion happens in the proximity of a strong promotor for human cells.
So, you better check that your vector has a promotor. I'm not sure if T/A vectors with a strong promotor exist, I myself am using a TOPO-blunt directional cloning vector (from invitrogen).
vairus is right. TA cloning is easier. But appart if it's for amplify the TA plasmid and directly cut the fragment (easier than multiples PCR) why don't you directly clone in the final plasmid ? What's your purpose with this fragment?
I think you are asking if it's OK to express DNA from one cell type in another?
If they're both human cell lines, they have the same genes already...remember?...you just may change the genotype and certainly the expression pattern
How much expression do you need in the new line? Are you trying to massively overproduce your protein?
Aside from just having a promoter, your vector's promoter should be maybe inducible if at all possible so you can turn it on and off.
First of all, thanks you all for your assistance.
Various: - For TA cloning I'm thinking to use pcDNA3.1/V5-HIS-lacZ (maybe without lacZ) which includes CMV as a promoter.
Fred_33: My final ideas is as you suggest, directly clone the sequence in the final plasmid using TA cloning. Briefly, my porpose with this fragment is to get the protein expressed in order to discover how it assemble with other proteins in the cell membrane. After doing this first step, I will do the same but joining it to GFP protein. Finally, I will study the differences.
Aimikins: You are right about what I want with the different cell lines.
- My idea is to start with normal expression. Depending on the results obtained, then maybe I will need to overexpress it.
- I guess that if I use different cell lines, I can't study the up/down regulation using Real-Time PCR because the results might be different due to the genotype and the expression pattern as you say, am I right?
- About "my vector's promoter should be maybe inducible if at all possible so you can turn it on and off", I haven't a clue if the one I will use has that characteristic or not.
I hope I make it more clear this time.
Thank you again. It's been really helpful. I really don't want anything to go wrong.
i think you'll probably save time and some money by directly precipitate your PCR product / Digest it by appropriate enzymes / cloe it in your final plasmid.
If you need to make a fusion protein with GFP after this protein, you'll just need to cut your vector...
But if you have tried to insert your PCR fragment during a long time wihtout success, yep topo cloning kit will save your time.