With PCR, it is supposed to get two bands of PCR products one is 258 bp and another is 222 bp. Anyhow, with 2% gel, only one bold band could be seen(>200 bp) and if run it longer, only smear could be seen. Any good suggestion?
They could be separated with 2% gel clearly when the thinnest electrophoresis pore was selected and the Goldview dye was empoiled. Good luck..
Usually, to prevent heteroduplex formation, I use in thermal treatment before gel electrophoresis. This treatment includes heating to 95C and slow cooling to room temperature. In order to separate the similar length fragments (2-10% differences) I use in 3-4% gel of MetaPhore or SFR (SuperFineResolution) agaroses.
The fastest: Why don't you use labelled primers. Don't know, how expensive labelled primers are, but a capillary seqencing run costs 5 €/CHF/$ (outsourced to company) ...
€dit: just to be sure, my answer was not too short and/or to cryptic: see attached picture/link to get an idea, how length polymorphism detection with a capillary sequencer works /€
The cheapest: might be for sure PAGE.
https://wwwnc.cdc.gov/eid/images/04-0875-F3.gif
All above mentioned are good and proposed strategies should work. Just a note: The conventional agarose gel electrophoresis should be carried out on relatively long gels. The longer DNA migration path results in a better separation of fragments. Ideally, 10-15 cm gels are OK.
hi, maybe some of your colleges have the following instrument (or similar from other companies) https://www.qiagen.com/us/shop/automated-solutions/quality-assurance/qiaxcel-advanced-system/.
This machine gives you a very fine resolution of the expected bands.
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