Hello,

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.

Its must be problem with the buffer. Take from other lab and just run it. Make sure the current applied is perfect with voltage. 1x buffer for gel and 1x for the electrophoresis. I happens same when I added the wrong buffer conc for gel making. No matter you use EtBr in gel or at post electrophoresis step if gel is problem then shifting EtBr--- no need.
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.
good luck

what is the expected size of your amplicon? did you tried using online tools to calculate the Tm for PCR. If not then please just check on one of the following and make sure it is 2-5 C less that the Basic Tm calculated.

Hello to everyone. I've been having problem with electrophoresis the past 2 weeks. A white cloud appears in more than upper half of the gel as shown in the picture. I usually make 2% agarose gel with TAE 50x. I make a stock of 1500 mL andI use 50 ml of this solution for the gel, with 3ul of Etrb (10mg/mL). I load the wells with 6ul (5uL dna and 1 uL of dye) For the ladder I use 1 uL of dye and 2 uL of ladder. I dont know what the hell is going on. I decreased the load of Etrb to 3uL ( before I use 5uL), I bought a new dye but the problem persists, also tried changing exposure times in UV transilluminator. I read this could be due to RNA contamination? Because sometimes they use this table to get RNA also. Suggestions will be greatly appreaciated. Thank you very much.