I have clone genes now i am making dsRNA to inject the specific gene to insect body for gene expression. Now i have Plasmid of all genes i run simple PCR with that plasmid use as template after that i use PCR Product from plasmid. I dilute 1:49 total 50ul of that PCR product 1ul PCR product and 49ul auto claved water ddh20 and run PCR with primers which i already design for gene expression using T7 Promoter. After i get bands restore DNA with AXYGEN Kit.
DNA precipitation : use the DNA which get from PCR with Promoter Primers for precipitation. Make Sodium acetate 3M with 5.2Ph using autoclaved dd water also auto clave sodium acetate. make 100% Ethanol(C2H6O) and 70% Ethanol keep in -20 before use. Combine DNA 100ul+10ul Sodium acetate mix well and put 300ul ethanol again mix well and keep in -20. After 1 hour centrifuge @ 12000RPM for 20 minutes but unfortunately i have no DNA precipitate????
any person guide me how to get pellets of DNA????
thanks in advance.
Hello Saad,
DNA pellets are difficult to see if they contain less than ~ 1 µg DNA. Unless incompatible with your further experiments, try adding ~ 5-10 µg glycogen (molecular biology grade) as a carrier. Otherwise, be sure to suck off the supernatant of your precipitate extremely gently in order to avoid discarding the pellet at the same time.
Hello Saad,
DNA pellets are difficult to see if they contain less than ~ 1 µg DNA. Unless incompatible with your further experiments, try adding ~ 5-10 µg glycogen (molecular biology grade) as a carrier. Otherwise, be sure to suck off the supernatant of your precipitate extremely gently in order to avoid discarding the pellet at the same time.
Have you tried checking DNA concentration before and after precipitation. So you would have an idea that DNA are there before precipitation. Precipitates might not be visible but when checking the concentration, DNA is still there. Or try tubes with filter some kits have those kind of tubes so pellets might not be discarded if that is the problem.
hi
i would recommend you to proceed further with the same sample. i understand that your starting material is PCR product..
Though you are not seeing any precipitate, the DNA will be there inside the tube for sure..Confirm this by checking an aliquot of the sample in agarose gel
In my experience, when we use ethanol for precipitation, the pellet will be rather invisible but the DNA will be there . and when we used isopropanol for precipitation of DNA, then there is more chance of seeing a pellet (even small)
But all these depends on the starting material..for genomic DNA isolation/plasmid isolation, you are starting with large amount of material , so there will be copious amount of DNA that will make a quite visible pellet after precipitation..
Your DNA is 100 ul add 200 ul of 100% ethanol plus 10% of sodium acetate (which is 10 ul) of original volume of DNA. That going to work. You will see a thin film on the side of well. Just rinse with 70% ethanol do not dry completely.Dry DNA does not dissolve properly.
...Instead of glycogen, helpful as recommended by Pierre but expensive, you can use 5 µg of linear acrylamide which can be prepared rapidly in amounts enough for decades. This fixe your precipitated DNA at the tube bottom, forms a visible pellet and is neutral in all Downstream applications.
If you are interested, I can supply a protocol for the preparation.
Best,
Günther
Hi Günther, Could you please send the protocol of preparation of 5 µg of linear acrylamide
.
Hi!!! You can follow the next step.
Add 0.1 volumen of sodium acetate 3M pH 5.2 and 2 volumen of ethanol 100% , and mix gently
Incubate at -20 C for 20 minutes.
Collect the precipitated nucleic acids by centrifugation at 13000 RPM (maximum speed) for 15 minutes.
Remove the supernatant by gentle aspiration. (it can apper a white pellet )
Add 0.5 ml of ethanol to the pellet and invert the closed tube sevveral time. Recover the DNA by centrifugation at maximum speed for 5 minutes in temperature room.
Remove all the supernatant by gentle aspiration. Use a kimwipe or disposable pipette tip to remove any drops of fluid adhering to the walls of the tube
Dissilve the nucleic acid gently in the same initial volumen or that you want.
In some cases, you can see the pellet (white), but if you cannot, dont worry, you follow the next step.
And finally, You should check the DNA by gel electrophoresis
I hope that this protocol will be useful for you, because I use it all the time without matter the concentration in the sample.
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