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small PCR products on agarose gel? - (Apr/02/2009 )

hi folks,
please help me with this:

I'm running a PCR with ca. 50-100 bp products
I have a high resoluton agarose suitable for small pcr frags (from merck)
The question is - what percentage of the gel should I use to obtain best separation?
best regards

Michal

-kpt.kliper-

Hey,

From ABI, this is what they recommend:

1.8% gel for 5001000 bp
3.0% gel for 150600 bp
4.5% gel for 30350 bp

However, it varies from company to company (somehow it is different), so do check out the information sheet that comes with it.

best,
TC

-T C-

Fermentas recommend

3% gel for 25-1000 bp
4% gel for 10-500 bp
5% gel for 10-300 bp

TIP- If you want to do a 3% gel or higher, let the agarose sit in the buffer (TAE or TBE) for at least 15 minutes before you heat it.

-molgen-

Hey,

Just wondering how this helps?

TC

molgen on Apr 2 2009, 06:17 PM said:

Fermentas recommend

TIP- If you want to do a 3% gel or higher, let the agarose sit in the buffer (TAE or TBE) for at least 15 minutes before you heat it.

-T C-

T C on Apr 2 2009, 02:29 PM said:

Hey,

Just wondering how this helps?

TC

molgen on Apr 2 2009, 06:17 PM said:

Fermentas recommend

TIP- If you want to do a 3% gel or higher, let the agarose sit in the buffer (TAE or TBE) for at least 15 minutes before you heat it.



The agarose gets hydrated and melts better. Otherwise you can get a gel where some of the agarose hasn't melted completely. When you run the gel it looks like dots or specks that hinder the bands from running correctly. The bands sometimes look a bit like an upside down "V"

-molgen-

hey
thanks for the tips people!
unfortunately the data sheet doesn't say anything about the recommended gel percentage...
I'm going to prepare the gel tomorrow so I will let you know if it helped or not :)
regards
Michal

-kpt.kliper-

For small fragments, you may find that a PAGE gel works as well or better.

-phage434-

well,
I think the soaking tip worked, I had no problems with pouring 4% agarose, no problems with air bubbles either (actually I dcant tell how it would behave without the soaking though!:rolleyes:)
The 4% agarose behaved like a 1% one during heating and pouring, which I must say - surprised me
I do have a problem with high background though when I'm trying to take a photo of the gel. The amount of the product is low and I have to use high exposure times to be able to visualize product bands...any tips on that people?
thanx
Michal

-kpt.kliper-

kpt.kliper on Apr 3 2009, 03:37 PM said:

I do have a problem with high background though when I'm trying to take a photo of the gel. The amount of the product is low and I have to use high exposure times to be able to visualize product bands...any tips on that people?


You could use your pcr fragment again as a template for another round of pcr. This should result in higher amplification yield.

-Sciurus-