Is my technique at fault while loading gels for electrophoresis?

18 December 2018 4 1K Report

I recently completed a gradient PCR but my bands were much less than satisfactory. I have a nagging feeling that my pipetting technique is at fault when loading. I use standard forward pipetting usually (Take up solution with 1st stop and expel with 2nd stop).

However, sometimes when I place my pipette into the well I can see the sample in my tip rise UP the pipette tip and when I attempt to pipette it out, some remains in tip (even after going to the 2nd stop). After this, there is visibly less sample in the well.

The samples are mixed with the orange G loading dye.

Admittedly, my poor bands could be due to a number of steps during the process (or simply the fact that PCR is tempermental).

Thank you very much in advance for all your advice!

Miaad K. Alkhudhairy Popular answer

Hi Robert flynn

Please see the attached link:

https://handling-solutions.eppendorf.com/liquid-handling/pipetting-facts/small-volumes/detailview/news/how-to-pipette-small-volumes-with-handheld-manual-pipettes/

Maurice Ekpenyong

If you identified these possible sources of errors, why don't you correct them? Alternatively, why don't you simply follow standard methods for loading gels if yours is giving you problems.

Add loading buffer to each of your DNA samples.

Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and allows you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases the density of your DNA sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer.

Once solidified, place the agarose gel into the gel box (electrophoresis unit).

Fill gel box with 1xTAE (or TBE) until the gel is covered.

Quick-Tip* Remember, if you added EtBr to your gel, add some to the buffer as well. EtBr is positively charged and will run the opposite direction from the DNA. So if you run the gel without EtBr in the buffer you will reach a point where the DNA will be in the bottom portion of the gel, but all of the EtBr will be in the top portion and your bands will be differentially intense. If this happens, you can just soak the gel in EtBr solution and rinse with water to even out the staining after the gel has been run, just as you would if you had not added EtBr to the gel in the first place.

Carefully load a molecular weight ladder into the first lane of the gel. When loading the sample in the well, maintain positive pressure on the sample to prevent bubbles or buffer from entering the tip. Place the very top of the tip of the pipette into the buffer just above the well. Very slowly and steadily, push the sample out and watch as the sample fills the well. After all of the sample is unloaded, push the pipettor to the second stop and carefully raise the pipette straight out of the buffer.

Carefully load your samples into the additional wells of the gel.

Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage.

Note: Black is negative, red is positive. The DNA is negatively charged and will run towards the positive electrode. Always Run to Red.

Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.

(Optional) If you did not add EtBr to the gel and buffer, place the gel into a container filled with 100 mL of TAE running buffer and 5 μL of EtBr, place on a rocker for 20-30 mins, replace EtBr solution with water and destain for 5 mins.

Using any device that has UV light, visualize your DNA fragments. The fragments of DNA are usually referred to as ‘bands’ due to their appearance on the gel

John Hildyard

"Much less than satisfactory" is frustratingly vague. If your bands are faint, that might be due to poor loading (i.e. most of your sample either wasn't loaded, or bubbled out of the well). If your bands are streaky/blurry/not the right size, that is not something you can rightly ascribe to poor pipetting technique. Personally, I find it difficult to believe you could be pipetting so badly that it is affecting your data: the most likely culprit is your actual PCR (gradient PCRs can be tricky).

Miaad K. Alkhudhairy

Hi Robert flynn

Please see the attached link:

https://handling-solutions.eppendorf.com/liquid-handling/pipetting-facts/small-volumes/detailview/news/how-to-pipette-small-volumes-with-handheld-manual-pipettes/

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