Agarose gel electrophoresis troubleshooting
Posted 08 August 2011 - 11:15 PM
I have to do my primer walking on one gene (length of my cDNA 3000bp). Whole gene is covered with 11 amplicons approx. 300bp. For my primer walking I choose forward primer from amplicon 1. and reverse primer from amplicon 11.
I did my PCR with one of the lowes temperature (I made my choice by looking at the temperatures from other amplicons on that gene).
And when I put my sample on gel I got no band. I use o,8% agaraose gel in TBE buffer (everything fresh and new). I also made my PCR reaction in volume 50ul, and after PCR I add 10ul of loadding buffer.
Any ideas, why I got no bands?
Thanks a lot for your help.
Posted 09 April 2012 - 06:10 AM
Make sure that the buffer you made is of correct composition, some time mistake happens which you may not be able to figure if out.
Posted 09 April 2012 - 06:16 AM
Posted 16 April 2012 - 03:37 PM
Posted 07 May 2012 - 03:13 PM
Posted 31 May 2012 - 09:20 PM
Posted 01 June 2012 - 05:46 PM
Posted 13 August 2012 - 07:29 AM
1 - EtBr diffuses out of the gel into the running buffer
2 - EtBr migrates upstream toward cathode, in opposite direction of DNA).
When not in use, store in the dark everything that contains EtBr (EtBr stocks, gels and buffers).
When not in use cover our gels and electrophoresis tank with a heavy dark plastic shopping basket (large grocers sell these).
If there is still some gel remaining we cover the gel/tank assembly with the shopping basket on a cart, and and store in walk-in fridge.