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.

helena kyle

Member Since 13 Oct 2012
Offline Last Active Dec 17 2012 08:42 PM

Find content

Community Stats

Group Members
Active Posts 4
Profile Views 173
Member Title member
Age Age Unknown
Birthday Birthday Unknown
Gender
Not Telling

About me

My research interests
yeast,cloning,expression, knock-out

Contact Information

0 Neutral

Friends

helena kyle hasn't added any friends yet.

Latest Visitors

helpwithdna
16 Dec 2012 - 15:16

#146782 cloning pcr insert into plasmid

Posted bob1 on 16 December 2012 - 01:24 AM

Yes, it is possible that 2 bp is too few. However, NEB says that 2 should be enough... Try using an excess of RE relative to amount of DNA. You do need to dephosphorylate your vector.

DNA ratios for cloning need to be molar ratios. Calculated as follows: ng insert = ratio x [insert length (bp)/vector length (bp)] x ng vector.

Make sure that the amount of ligation mix used in the transformation is less than 5% of the volume of the transformation volume.

#143521 colony cracking for screening of positive clones

Posted leelee on 17 October 2012 - 12:11 AM

The pink line is the band for genomic DNA

Probably yes. This is DNA that is still trapped in the well, so likely to be genomic.

The blue line is where two bands were observed would be the RNA (Is the faint band across the lane is RNA?)

I would say nope. I very much doubt that RNA would come out nice clean bands like this, I would say these are more likely your plasmid in two separate conformations.
The RNA is likely to be the smear at the bottom that you have marked with the purple line.

The purple line is where the proteins were.

I wouldn't expect to see protein at all with AGE....(and as I said, my guess is this is your RNA)

The yellow line is the PLASMID.

agreed

As for which bands to compare, I can't tell you as I've not used this method before... sorry Posted Image