I am running the vertical gel electrophoresis for separating fragments in the PCR products digested with restriction enzyme. After running, the gel has to be separated from the glasses of the tank ready to be stained for visualization. The problem is that, the gel sticks completely to the glasses and when extreme force is used the gel become distorted. What can I do to overcome this problem?
Clean EVERYTHING with ethanol and then rinse it with distilled water, apply on one glass a silicon repellent (rain-x for instance) and clean it lightly with ethanol 70% (I usually apply it on the short glass). Mark the glass you treat as the gel will detach from it!
wash the plates in cool running buffer.Then if you are using Bio-Rads gel apparatus, they provide you with a thin plastic strip (green in colour). you can insert this in between your plates and slowly try to separate the plates. If you dont have the plastic strip, you can also substitute it with any flat thin plastic strip, and do the same. The magic is to spread the force evenly on the plates when you force the plates separate. Practice makes perfect!
Clean EVERYTHING with ethanol and then rinse it with distilled water, apply on one glass a silicon repellent (rain-x for instance) and clean it lightly with ethanol 70% (I usually apply it on the short glass). Mark the glass you treat as the gel will detach from it!
If you are having to use extreme force, make sure that the glass plates you are using have not previously been treated with an agent such as "Glass Bond". This is designed to make your gels stick to one of your plates. If both plates are coated with this it would require a great deal of force to separate them.
Yes, Steve is right! Glass bond is used when you need to dry the gel on the glass and expose it. If that's the case, you need to soak the plates in hot water and soap for a while and clean them thoroughly with acetone a couple of times. After that start with etOH and use the silicone repellent!
I've had problems with low % native bis-tris gels in the past. I found that treating the glass plates with sigmacote - an easy silicone treatment - and lifting the gel off under buffer or water did the trick. We used to do the same thing for sequencing gels......
In addition to a good cleaning as suggested, I would just take out one plate and soak the gel stuck to the other plate into the staining solution tank. It comes out easily. If your gel is 5% then it will be very fragile....
After washing and cleaning the glass plates you soak them with 1% SDS and let them dry by standing vertical on paper towels.
I agree with Ruben. This method is easy and does not harm the electrophoresis run.
Two methods for making the glass non-stick:
1) The already-mentioned RainX (from the auto department of your local discount store). (I started using it for my sequencing gels but soon learned to also love it where it was designed to go: my windshield.)
2) Dichlorodimethyl silane. Make a fresh dilute solution (I don't remember the details; I think it was either in absolute EtOH or in CHCl3. Do NOT use water.) and rub well with lab tissue onto a very clean, dry glass surface. Then rinse well with EtOH to remove excess reagent and H2O to remove EtOH. Hydroxyls on the glass surface displace the chlorine, forming a chemical bond. WARNING: This material is volatile and reactive; don't breath it or get it on your skin. You can also make other glass items, like the insides of pipets and beakers, hydrophobic the same way, by cleaning them well and then treating with the reagent, followed by EtOH and H2O rinsing.
RainX, basically wax, can be removed with detergents. Dimethylsilane, chemically bonded to the glass, can be removed by soaking the glass in strong alkali.
The plates absolutely must be clean. I used to have much success if I soaked the plates in RO water, with 1-2 fresh changes, for at least overnight.
If the gel is scanned from only one side (storm/typhoon, etc) I like this trick. Clean the plates really well and use rain-x, pam, whatever only on the inside of the front plate. This way this plate comes off really easily. Then use old x-ray film or thin layer plastic of any kind to stick the gel to. Then you can easily roll the gel-plastic off the second plate. You can still stain on the plastic (does take a little longer) and gives you a great way to move the gel around without damaging it. The only other down side is you can't dry it either (unless you buy special membranes to put the gel on).
The plates should be perfectly cleaned. Dry them and apply sigmacote along the edges of the front plate. The plates comes off easily. soak the gel stuck to the other plate into the staining solution tank. The gel comes out easily and further lifting/drying use X-ray film. It works for sequencing gels as well.
In our lab we overcome this problem even without carefull cleaning of rhe plates by using of Dichlorodimethysilane in 1,1,1 - Trichloroethene / Promega. We apply a small quantity on the shorter plate and clean with a filter papper. Then with the same paper rust remove the dust from the bigger plate.
Get a Tupperware-like container that fits the glass plates. Drill a hole in the lid and mount a hose adapter to it. Put the plate in with spacers so a air flow can occur around the useful area. Put a thimble size container in with the plates. Fill the container 1/4 full with Sigma Coat. Put the lid on, attach a hose and pull a moderate vacuum. Clamp the hose and let the Sigma Coat vapors do their magic. No worries!
Done this for 25 years.
I forgot to say that we leave the plateS in the container for about 12 hours. By placing spacers around them, all surfaces of both plates get coated.
Use two sets of gloves. Apply binding silane in the one plate and treat the other so that it doesn't stick.
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