Large DNA extraction from an agarose gel (View forum version)



rota

Posted 14 October 2010 - 02:29 PM

I'm trying to extract a lineal plasmid of 28kb from an agarose gel. I've tryed with the Qiagen kit but it didn't work because of the size of the plasmid.
I need it to do a ligation
any suggestions?
Thanks!

pDNA

Posted 15 October 2010 - 08:47 AM

the problem with large fragments and column based purification methods is that large fragments elute poorly from the column. Maybe there exists a work-around using agarase and e.g. isopropanol precipitation if column based methods do fail!

Regards,
p

perneseblue

Posted 18 October 2010 - 07:07 PM

if your lab the money, you could buy an eletrolution box. The devise would pull the DNA out of the gel and concentrate it in a small volume.

If you can't buy this kit, you can do electrolution using a dialysis tube, an electroporation box and if required butanol dehydration to concentrate the DNA.

hematopoietry

Posted 20 October 2010 - 01:00 AM

I'm trying to extract a lineal plasmid of 28kb from an agarose gel. I've tryed with the Qiagen kit but it didn't work because of the size of the plasmid.
I need it to do a ligation
any suggestions?
Thanks!


I would recommend:
-Use a bead-based purification method (JETSORB OR QIAEX II. It can purify fragments up to 50kb)
-Isopropanol precipitation of the DNA
-Alternatively, you could try and run the DNA on low-melt agarose gel. After running, you just melt the excised bands, add them together, add ligase+buffer+ATP and ligate like that.

phage434

Posted 20 October 2010 - 04:59 AM

You can also digest the agarose with beta-agarase (NEB).

rota

Posted 27 October 2010 - 05:32 AM

Thank you!!
The QIAEX II worked! :)))

mahsa

Posted 02 November 2010 - 02:03 AM

the problem with large fragments and column based purification methods is that large fragments elute poorly from the column. Maybe there exists a work-around using agarase and e.g. isopropanol precipitation if column based methods do fail!

Regards,
p


hey there P
when you say large, how large do you mean? I have difficulty purifying a 7.5 kb fragment from agarose gel using high pure pcr product purification kit( roche) which guarantees the purification of fragments of these sizes with the efficiency of ap.90( which I never have gotten so far). is it because of the size or I have to look for the culprit somewhere els?

best
mahsa

Curtis

Posted 02 November 2010 - 11:26 PM

I hate QIAEX II. That would be my last choice. It is precipitation based but never gave me enough DNA.

rota

Posted 16 November 2010 - 01:06 PM

and which would be your first choice?
thanks!