11 replies to this topic

[lactamase]

I tried Roche, promega, Bioneer, Qiagen.  I would say, Roche kit give the best yield


how about you guy ?

Edited by lactamase, 07 August 2009 - 01:06 AM.

[phage434]

Best approach: design your protocol to eliminate it.

[Andriy]

My favorite is GFX kit from GE Healthcar
.
Can't tell you how it goes with the yield comparing to other kits, but it's clear advantage is that it requires three spins instead of five: Only one spin to load (you add equal volume of capture buffer to dissolve agarose, not several volumes) and one spin with ethanol (again, only 500 ul of ethanol which is removed completely without additional centrifuging step)

Hey phage434, your idea is interesting, but how do you eliminate gel purification?

[appleyun]

Hi frens!

For my work, Roche kit gave low yield! I suggest INTRON kit (for high yield) and GeneAll (for high purity). Check this out!

Cheers,
appleyun

[fishdoc]

I've used Qiagen (minelute and qiaquick), an invitrogen kit, and a zymo kit.

Qiagen and invitrogen seemed pretty comparable. While using the zymo kit, a purification would yield no detectable DNA, but using the same conditions with Qiagen gave a pretty good amount, so zymo seemed pretty poor for me.

[HomeBrew]

For cloning, it's not so much the quantity of DNA as it is the quantity of undamaged DNA.  You can improve your cloning results dramatically by following the simple step of adding 0.28 g/L guanosine to 1x TAE as a UV protectant (stir with gentle heat until dissolved), and using this to cast and run your gel.  See here.

We use the Qiagen kit, and have had good results with it, but I think the results are influenced more by adding the guanosine than by the particular kit used.

[jiajia1987]

I like Qiagen and Zymo.

[hanming86]

Run two lane and the same time. split to half, one get EtBr and one doesn't.
use that as reference.

no UV yay!

[HomeBrew]

hanming86, on Aug 12 2009, 09:46 AM, said:

Run two lane and the same time. split to half, one get EtBr and one doesn't.

That requires twice as much sample.  Just make up 4L of 1x TAE + gaunosine for casting and running gels from which you're going to recover bands and be done with it...

[Qundo12]

HomeBrew, on Aug 13 2009, 06:08 AM, said:

hanming86, on Aug 12 2009, 09:46 AM, said:

Run two lane and the same time. split to half, one get EtBr and one doesn't.

That requires twice as much sample.  Just make up 4L of 1x TAE + gaunosine for casting and running gels from which you're going to recover bands and be done with it...

I used to do the method hamming86 mentioned, don't need much of sample, just some ul of extra sample to use as a control. For example: run 100 ul sample on the left lane + 10 ul sample on the right lane. After running, cut the gel into 2 parts, stain only the right one in EtBr, mark the location of the band by observing on UV lamp and cutting. Then place the two parts together, excise the large, unstained one based on the marker sites on the small one. Confirm the cut by staining the remaining gel. No EtBr, no UV ^^

Edited by Quasimondo, 12 August 2009 - 05:23 PM.

[HomeBrew]

I know that method works; it just seems simpler to use a buffer with a UV protectant in it.

BTW, when I do use that method, I just stain the whole gel, place a piece of paper in a sheet protector on the UV transilluminator, place the whole gel on the paper with one lane hanging over the edge (and thus exposed to the UV), and slice as needed.  It's easier and more accurate.

[Ivanov_br]

Qiagen and MN are quite good! Good results with Invitrogen too, but MN is cheaper!