Agarose gel problem.. plz help - (Apr/26/2009 )

Hello!
1)I've a 420 bp PCR fragment. On restriction enzyme digestion 2 fragments are produced- 386bp and 34 bp..
I tried running the RFLP products and the undigested PCR product (control) on 2 % agarose but i've failed to obtain a good resolution..Can any1 suggest wht percentage of agarose wud be appropriate to obtain a good resolution.
2)Also when i load my RFLP samples it seems to run unevenly ( the samples form an arc) .Can this be due to excess sample loading.. I checked the buffer level..the gel is always immersed in the buffer (TBE). The voltage that i apply is 120. and yes the arc is obtained when i load the sampl in another appratus too.Plz help...

Hey,

Choose:

1. Digest with another enzyme which would atleast give you 100 bp fragment, 150 + 250 would be good.

2. 34 bp is tricky on agarose gel, try PAGE.

3. In my view 1.5% should do if u still wana go for agarose but its tricky.

4. The arc could be due to the tank, run it in another apparatus and check.

Hope it helps.

Best,
TC

Shruti B on Apr 26 2009, 11:10 AM said:

the arc is obtained even when i load the samples in another electrophoretic apparatus.. what shud I do? and i cannot use another restriction enzyme.

Oh yeah you write it in yr last post as well...I missed that....Its the bands which form a small arc (which could be due to high concentration and is okay) or its due to differential mobility in various wells? (thats what I gathered earlier and this is not okay).

Best,
TC

Shruti B on Apr 26 2009, 07:22 PM said:

differential mobility in different wells.. that is what is happening.. i tried in different apparatus.. but the same thing happened again.. the buffer is fine coz my colleague used it too and had no such problem...

So clearly its got something to do with yr sample. To confirm this, maybe you can run the sample (or the ladder) in all the wells (alternate wells will do) and check if you still see it, if not then there is somethign in yr sample that you need to take care of.

Best,
TC

Shruti B on Apr 26 2009, 11:28 PM said:

differential mobility in different wells.. that is what is happening.. i tried in different apparatus.. but the same thing happened again.. the buffer is fine coz my colleague used it too and had no such problem...

ok.. and correct me if i'm wrong but if i wanna increase the difference between 420 and 386 bp fragments then shudn't i be increasing the agarose % ? say 3% etc.

does the "arc" look like a smile?

smiling is caused by uneven heating of the gel. you may want to try running at a lower voltage.

if you can't resolve the 420 and 386 bp fragments then you can use a higher concentration or switch to "metaphor" agarose.

I agree 2% agarose with 120 V is perhaps too much, I'd try 80-100 for better results. Another way to increase resolution are longer gels. If you don't have a larger chamber, try to run the gel as far as possible (and stain it afterwards, if the EtBr is already gone).

... or you could run the gel in borate alone. Make up your gel in this buffer:

20x recipe: 38 g/l sodium borate
24 g/l boric acid

The gel will run much faster, so slow it down a bit. Also, without the Tris and EDTA, there is no effective buffering, so you can't re-use the buffer more than a couple of times. The good thing is that you should be able to resolve the difference between 420 and 386 bp. (I have separated the "500 bp" double band on a DNA ladder into the 504 and 517 bands, so I speak from experience). Also, if you are in a rush, the gel can run very fast without heating (up to a point, of course); the main culprit for heating is Tris.