Google Sign in options
Remember me
This is not recommended for shared computers

Sign in anonymously
Don't add me to the active users list

Privacy Policy
Sign In
Create Account

Javascript Disabled Detected

You currently have javascript disabled. Several functions may not work. Please re-enable javascript to access full functionality.

Labmouse9

Member Since 17 Jul 2012
Offline Last Active Apr 14 2013 04:28 PM

Find content

Community Stats

Group Active Members
Active Posts 5
Profile Views 399
Member Title member
Age 26 years old
Birthday November 29, 1986
Gender
Female
Location
Ontario

About me

My research interests
Huntington's and other neurogenerative diseases

Contact Information

1 Neutral

Friends

Labmouse9 hasn't added any friends yet.

Latest Visitors

lab rat
10 Nov 2012 - 17:29
Curtis
17 Jul 2012 - 21:21

Posts I've Made

In Topic: Primer Check?

18 July 2012 - 09:39 AM

Hey!
Thank you so much for your reply

I added the extra A to the mCer primer because I was changing it from a C1 vector to an N3 vector. I guess I was just confused! Thanks!!!

In Topic: PCR product smearing on gel

17 July 2012 - 04:31 PM

stagia, on 27 October 2011 - 11:41 AM, said:

hi
I had the same problem. I am trying to amplify a 1.6kb product using as template cDNA synthesized either with oligodT or with gene specific reverse primer. what I get is a band of correct size and a smear around it only from 1kb to 2.5kb. Have you any idea what I should do? I have tried 3 different polymerases and almost always the same picture
thanks
stagia

The smearing is a result of accumulation of enzymes extending unspecific targets. To prevent this, there is a number of things you can try!
You can decrease your enzyme concentrations (I usually find 0.5ul sufficient for Taq or Pfu), lowering Mg2+ concentrations as mentioned about. You may need to decrease extension time (each polymerase will give you a speed at which it extends DNA so you can adjust accordingly) or perhaps reducing the total number of cycles. You may also lengthen the denaturing time and or raise the denaturing temperature.
Andddd of course, it could be contamination of any of your reagents as discussed above.

In Topic: molecular cloning

17 July 2012 - 04:12 PM

For the double digest, when you run it on a gel, you'll have to run it with a 1kb ladder like the following: http://products.invi...roduct/10787018
This will come with a handout of how to interpret your gel.

If you find that your when you load/run your digests on a gel, the digests remain at the top and do not run (thus will appear higher than the ladder) this means the vector DNA has remained circular and did not cut. Otherwise, you can line up the digest products to the ladder to ensure their size.

It might also help to CIP the vector after digesting, which prevents recently digested vectors from closing on themselves, thus rendering them unable to include an insert. http://www.neb.com/n...roductM0289.asp
You may not need this but if you find once your plate your ligations that you have a lot of colonies on your control plate, this could help!