Agarose gel electrophoresis troubleshooting - (May/01/2009 )

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.

Hello to everyone. I had some problem whit electrophoresis and I don't know what's wrong. So... I'm working gene cloning and I isolated my plasmid (pet20b) from transformed cells. After that I digested my plasmids with BamHI (1h on 37 ⁰C ). After digestion, I worked agarose electrophoresis (on 80 V). Then I transfered my gel in EtBr (10 minuts). After photography gel in GelDoc, I don't had any bands, no bands from my samples, no bands from my markers Why? What you mean about this? Are you had the same problem? ( I did everything right, by protocols... I don't know where my bands and what happened to them). Please, help me . Thank you for you opinion.



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Hello! I've been reading other comments about problems with visualizing DNA bands in agarose gels when the EtBr runs out of the gel (referring to adding EtBr directly to the molten agarose rather than doing post-electrophoresis staining and de-staining) . I'm wondering why it matters if the EtBr runs out of the gel, because EtBr is an intercalating agent. Shouldn't enough EtBr be stuck in the DNA so even if the rest of the EtBr runs out the top end of the gel the DNA bands will still be visible? Greatly appreciate any wisdom on this topic!

The EtBr does intercalate, but the amount that remains intercalated is less, so small and/or faint bands will not show up well.

Add EtBr to the running bubffer because:
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.