Need help with TOPO TA cloning problem! - TOPO TA clonig, ligation, no insert (Oct/02/2007 )
Well, my first suggestion right now is to not do PCR to screen your clones. For now, depend on the EcoRI digest to look for your gene. I think your PCR conditions are suspect and digests are faster anyway 
You said that the digests you see are only of plasmid, but you mentioned coiled and supercoiled plasmid. Are you sure they are? Does your DNA ladder show markers that will tell you what the MW of your bands are?
On a PCR gel picture you posted, it looked like there was a weak band in the first lane.. was that phantom when you digested?
To be sure, your Topo kit is not expired right? I've actually used a 1+ year old TopoTA kit and it worked but efficiency is much less.
I hope you get this clone soon!!
-Judes-
QUOTE (smu2 @ Jan 22 2008, 02:16 PM)
Hi Science Mama,
I just looked at the date of your original post - 3 months working on something as simple as a TOPO cloning is a LONG time! Is there anyone in your lab helping you with this? A graduate student or a post doc? If not, I would suggest that you get someone with some experience cloning to help you. If you are really stuck on this (and its only a first step for a bigger project), then I would probably recommend that you ask someone to do it with you side by side so that you can at least get past this part of the project and move on to the next step. As an undergrad, you're there to learn how science works but you also want to get the experience of having some success while you're doing it. Otherwise you'll come out of your internship thinking that science is too hard, boring, and that you don't want to pursue a career in this field.
- Just my 2 cents. Good luck to you.
I just looked at the date of your original post - 3 months working on something as simple as a TOPO cloning is a LONG time! Is there anyone in your lab helping you with this? A graduate student or a post doc? If not, I would suggest that you get someone with some experience cloning to help you. If you are really stuck on this (and its only a first step for a bigger project), then I would probably recommend that you ask someone to do it with you side by side so that you can at least get past this part of the project and move on to the next step. As an undergrad, you're there to learn how science works but you also want to get the experience of having some success while you're doing it. Otherwise you'll come out of your internship thinking that science is too hard, boring, and that you don't want to pursue a career in this field.
- Just my 2 cents. Good luck to you.
Oh, it's been longer than three months. My mentor, who has successfully cloned several types of HPV with the same kit, actually did the entire experiment through to completion several times with me only to find the same results I have been finding. He's the one who's been coordinating all the troubleshooting, and even he is at a loss. I know that science has its ups and downs, and he's assigned me other projects with real time PCR that have worked out perfectly, so we know I'm not some sort of jynx or something. I'm definitely pursuing a career in this field despite all the difficulties and frustration. Thanks!
-Science Mama-
QUOTE (Judes @ Jan 22 2008, 09:37 PM)
Well, my first suggestion right now is to not do PCR to screen your clones. For now, depend on the EcoRI digest to look for your gene. I think your PCR conditions are suspect and digests are faster anyway
You said that the digests you see are only of plasmid, but you mentioned coiled and supercoiled plasmid. Are you sure they are? Does your DNA ladder show markers that will tell you what the MW of your bands are?
On a PCR gel picture you posted, it looked like there was a weak band in the first lane.. was that phantom when you digested?
To be sure, your Topo kit is not expired right? I've actually used a 1+ year old TopoTA kit and it worked but efficiency is much less.
I hope you get this clone soon!!
You said that the digests you see are only of plasmid, but you mentioned coiled and supercoiled plasmid. Are you sure they are? Does your DNA ladder show markers that will tell you what the MW of your bands are?
On a PCR gel picture you posted, it looked like there was a weak band in the first lane.. was that phantom when you digested?
To be sure, your Topo kit is not expired right? I've actually used a 1+ year old TopoTA kit and it worked but efficiency is much less.
I hope you get this clone soon!!
As for being sure that the two bands that show up on all the gels after either PCR of plasmid prep or a digest are coiled and supercoils of the plasmid... that's what my mentor explained it was to me.
The picture of the gel post plasmid prep and pcr is just that. I did not digest that one. I'm not too sure what you mean by phantom.
Also, we have checked expiration dates on everything as soon as we started having trouble.
We've called invitrogen for troubleshooting with the kit before.... But for now, my mentor wants me to move onto another project involving real time pcr and then come back to this migraine later on.
Thanks all.
-Science Mama-
QUOTE (Science Mama @ Jan 23 2008, 09:40 AM)
Oh, it's been longer than three months. My mentor, who has successfully cloned several types of HPV with the same kit, actually did the entire experiment through to completion several times with me only to find the same results I have been finding. He's the one who's been coordinating all the troubleshooting, and even he is at a loss. I know that science has its ups and downs, and he's assigned me other projects with real time PCR that have worked out perfectly, so we know I'm not some sort of jynx or something. I'm definitely pursuing a career in this field despite all the difficulties and frustration. Thanks!
I'm glad to know that you're not on your own with this project - had me worried there for a while. And its good to hear that you're still enthusiastic about science. Keep up the good work!
-smu2-