pEGFP-C1

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderators: honeev, Leonid, amiradm, BioTeam

Post Reply
dky9829
Garter
Garter
Posts: 2
Joined: Fri Oct 28, 2011 3:10 am

pEGFP-C1

Post by dky9829 » Fri Oct 28, 2011 3:54 am

hello,I am a new one, I have a big problem in constructing vector. I have a muscle specific expression promoter,and I do double digestion in this specific promoter and pEGFP-c1 at the same time, then link the specific promoter into pEGFP-C1, replace the cmv. the problem is after transfection NO GFP expressed! WHY this happen? which step is wrong? help me!thanks!

daniel.kurz
Coral
Coral
Posts: 167
Joined: Fri May 19, 2006 1:47 am

Post by daniel.kurz » Fri Oct 28, 2011 5:53 am

Are you running a gel of your DNA after you do the ligation to confirm that you have your DNA fragment in the plasmid? It could be a question of your PCR or your transfection. I suspect that it is your PCR because normally if there was at least partial inclusion there would be some level of expression. Why are you just cloning in GFP alone not linked to a protein? Can you elaborate on the steps that you took so that there is a more clear picture?

dky9829
Garter
Garter
Posts: 2
Joined: Fri Oct 28, 2011 3:10 am

Re: pEGFP-C1

Post by dky9829 » Fri Oct 28, 2011 8:15 am

thanks your help! I have do PCR and sequencing to check if the specific promoter have been ligated into pEGFP-C1. I construct this vector for checking the activation of the specific promoter. now I will transfect cell again, I hope it will be ok! thanks

User avatar
JackBean
Inland Taipan
Inland Taipan
Posts: 5694
Joined: Mon Sep 14, 2009 7:12 pm

Post by JackBean » Mon Oct 31, 2011 12:46 pm

I would suspect the expression as being faulty. Are you sure you have whole promotor? Does it need any other elements?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

genetherapy
Garter
Garter
Posts: 33
Joined: Fri Nov 11, 2011 9:54 am

Post by genetherapy » Sat Nov 19, 2011 8:53 am

Hi,
I am going to construct a vector and will use GFP,too to see if my promoter and the gene that ı insert works so I'm very intrigued about this topic. Daniel asks "Why are you just cloning in GFP alone not linked to a protein?". What's that advantage,could you please explain?
I wish safe arival to you pEGFP-C1

daniel.kurz
Coral
Coral
Posts: 167
Joined: Fri May 19, 2006 1:47 am

Re:

Post by daniel.kurz » Mon Nov 21, 2011 2:39 am

genetherapy wrote:Hi,
I am going to construct a vector and will use GFP,too to see if my promoter and the gene that ı insert works so I'm very intrigued about this topic. Daniel asks "Why are you just cloning in GFP alone not linked to a protein?". What's that advantage,could you please explain?
I wish safe arival to you pEGFP-C1


Simply, easier to track the mistake that is being made if it doesn't ligate. Double digestion can lead to having an alternate sight other than the one that you want to cut at be used. You can accidentally drop out part of the gene that you want.

daniel.kurz
Coral
Coral
Posts: 167
Joined: Fri May 19, 2006 1:47 am

Re: pEGFP-C1

Post by daniel.kurz » Mon Nov 21, 2011 2:41 am

dky9829 wrote:hello,I am a new one, I have a big problem in constructing vector. I have a muscle specific expression promoter,and I do double digestion in this specific promoter and pEGFP-c1 at the same time, then link the specific promoter into pEGFP-C1, replace the cmv. the problem is after transfection NO GFP expressed! WHY this happen? which step is wrong? help me!thanks!


Someone in my lab has been having the same problem, she found out that one of her enzymes was cutting twice in her plasmid and was accidentally dropping part out. She says to not use double restriction cutting. She was able to resolve her problem over night after months of subcloning.

Post Reply

Who is online

Users browsing this forum: No registered users and 2 guests