Cloning - (Mar/21/2007 )

Hi,

I am trying to seperate the 35kb lambda DNA fragement in 0.5% agarose gel , it didn't work fine with QIAGEN kit . Can anyone suggest how to overcome the problem to get nearly 80% recovery or if I can do PCR for 35kb DNA fragement as template which enzyme I can use for the reaction. If anybody done it before pls suggest me.

Your responses are most appreaciated.


Thanks

With regards
Vishnu Reddy (Phd Student)
Viikki Biocampus
University of Helsinki

try electrolution, followed by butanol dehyrdation. You should be able to recover your large fragment without problem.

As for PCR of a 35kb fragment, it is doable but I would not advice it. Optimising the conditions for a PCR that long will take time, certainly longer then an afternoon doing an electrolution.

(You will need dialysis tubing with cut off point around 12kD to 14kD)

We purify 50 kb fragments with QIAEX II kit (for 40pb-50 kb) with very good recovery. I have done electroelution but I have better result with the kit.
Which kit are you using? QIAquick (this is for 100 pb-10 kb)?
If you are using QIAEXII:
Be sure wash solution has ethanol already.
Add 3v of Buffer QX1 plus 2v H20
Incubate 10 minutes at 50°C when eluting.

Hi,


Thanks for your reply. What is the initial conc. of the DNA? and how much did u load in agarose wells? How did u run the agarose gel like in hours and volts? did anything in the wells?

Pls reply.

Thanks in advance.

With cheers
Vishnu

Hi,

Sorry to mention I used the same kit what u said.

Cheers
Vishnu

Thanks for your reply. What is the initial conc. of the DNA? and how much did u load in agarose wells? How did u run the agarose gel like in hours and volts? did anything in the wells?

Depending what I’m doing or how much DNA I have. To purify, I run from 50 ng to 1 ug per well (7 x 2 mm), some times I join 2 or 3 well in the same tube when purifying.
If I’m purifying, I run a 0.5-0.7-agarosa gel at 50-60 V per 1.5-2.5 h. If I need a nice gel for the picture or want to have a good bands separation, I run a 0.4 agarosa gel at 25 V for many hours (10-15 h).
If you add SDS to the loading buffer and or heat the samples before loading, you’ll have a better separation.

And about QIAEXII kit. Ones, we had problems because the ethanol in the wash solution was evaporated a lot.
Read very carefully the user manual, because there are some special indications for large fragments purification like heat at 50°C for 10 minutes when eluting. Check the pH of your elution buffer (TE, Tris, etc).


I hope it could be helpful to you!!
Good luck.