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March 31, 2011 at 6:59 am #6338
Ran PCR reaction 50ul volume – the master mix had go taq green with my primers, ran 2% low/high 1XTAE agarose gel for the PCR product, got my desired band and a very clean single band at 511bp at that. Then I cut out the band and used “QIAquick Gel Extraction Kit” then took 2ul of purified DNA product into a 10ul total volume with loading buffer and ran the same gel to quantify. Ran 10ul of the pre-cleaned (ctrl)PCR product along side the cleaned up sample for comparison and yielded absolutely no DNA for the cleaned sample using the kit. (Brand new kit) and the the pre cleaned product that I ran along side as a ctrl showed exactly the same position as before, a nice 511bp band. Any ideas as to why I’m not getting my DNA band after clean up? I have used Qiaex II twice and now the third time I have used QiaQuik.
Three times but no cigar. Please help somebody!March 31, 2011 at 12:43 pm #18060
Did you check the kit’s various solutions? Some may need to have ethanol added before using them. Otherwise, warming the elution solution may increase the yield. Also, letting the melted gel and binding buffer incubate for 1-2 min on the column before spinning may help a bit, as well as letting the elution solution incubate for 1-2 min.
I’m also a bit vary about you only having 10 ul. Did you only elute in 10 ul? For many kits, this is not recommended (20-50 ul is usually a good range).
My experience with these types of kits is that the yield is very, very low and if you load onlu 2 ul on a gel you will perhaps not see it. Better measure with nanodrop, for instance.
I did 6 gel purifications with a kit from fermentas the other day and got 0.5 to 10 ng/ul in 50 ul elutions, which would definitely not be seen on a gel. However, that’s enough for me to proceed with ligation. If I need more DNA I never use kits, I instead use low melting agarose, cut the band, and digest the agarose with an enzyme called Agarase, followed by precipitation with ammonium acetate and ethanol.March 31, 2011 at 2:20 pm #18061
All solutions were checked and the appropriate volume of Ethanol was added to PE. I set up 4 reactions of 50ul each ran the gel got my nice bands saved an aliquot of my PCR product to run with the purified sample I got from the gel extracted sample. Thats what I meant when I used 10 ul of the original PCR product and it revealed the correct band. It’s the kit purified band that shows nothing when I run a gel to quantify the amount I yielded. These are brand new kits. Is the the master mix with go taq green causing this. Or am I ok with the gtg. My objective is to sequence from the gel extraction band. Should I just try purifying or cleaning up the PCR product directly without purifying from gel? It is a very clean single band from the PCR.March 31, 2011 at 3:01 pm #18062
I’ve never used the polymerase that you are using, but it would really surprise me if that was the problem. Did you add the optional volume of isopropanol before you bound the DNA to the column?
If you want to sequence this directly, I think you have to gel purify it. If you were cloning it, you might be able to get away with just a clean-up kit since you can screen out any non-511bp inserts that show up. If you happen to have a TA cloning kit in the lab, this might not be a bad idea to try – you can then just sequence the resulting construct.March 31, 2011 at 3:02 pm #18063
Check with the manufacturer if they think its go taq green causing it.
For sequencing you don’t need much so if you can check if you have any dna with nanodrop or something similarly sensitive you could perhaps sequence from the ppurifies product anyway.
As you say, since your band is nice you don’t have to go through the whole gel purification step. Theres a nice quick (and cheap) method with a enzyme called ExoSap-IT that’s excellent for preparing samples for sequencing…March 31, 2011 at 3:08 pm #18064
Another thought – the chaotrope that they use to dissolve the agarose is very powerful and will eventually denature the DNA. Make sure when you are using the kit that you don’t incubate the gel slice with the dissolving buffer longer than you need to. Personally, I don’t even use the 50°C conditions that the instruction call for – I just take the gel slice (cut up into ~10 peices to speed things up) and dissolving buffer and put it on a rotating wheel at room temperature, and the agarose dissolves within 5 minutes or so.April 1, 2011 at 12:22 am #18065
I’ll give it a try thanks for your help.April 1, 2011 at 12:26 am #18066
Another good point and tip in regards to cutting it up (surface to volume area ratio) and the time on temp I’ll pay close attention to that since all our timers batteries are dead. Time to get some batteries. But if any thing if I went over It would only be like 1-2 minutes. Will this denature DNA? Thanks for your help Jode.April 1, 2011 at 2:41 am #18067
It probably wouldn’t have denatured the DNA so badly that you wouldn’t have gotten anything back, but if the conditions were right (heat block was a little warmer than it was supposed to be, the DNA is AT-rich, etc.) then maybe. I keep a close eye on mine and load them ont he column as soon as I can no longer see any agarose “lenses”.April 7, 2011 at 7:42 pm #18074
Before the elution step you might want to try letting the elution solution incubate on the membrane for ~15mins. This seems to increase the final DNA  in our lab.April 9, 2011 at 3:15 am #18046
do u really need to do gel purification? do u have 2 bands? if not i would do just prc clean up (much better recovery). if u need to just sequence the pcr product, u dont even need to purify it, the company usually offers the clean up for small fee.
recovery for gel purification is very low. couple suggestions: load as much DNA as u can (tape 2 teeth on the comb to make a big well). use lower % of agarose u can. always use fresh buffers and clean elfo unit. do the optional clean with QC buffer. put PE buffer on the column and wait 2-3min then spin down and repeat it 3times. elute in warm (60-70C) elution buffer.
this way i usually get 40% recoveryApril 10, 2011 at 1:01 am #18041
The same thing happened to me last week. My problem was the pH after solubilization. Qiagen says add 10 uL of 3 M NaOAc pH 5 if your buffer is purple (i.e. high pH). But, it turns out orange is also too high. They give a figure where their DNA yield goes from 95% (yea right) to 5% immediately at pH 7.5. So who’s to judge orange from too orange? I now just add in the NaOAc each time and I now regularly get about 40-50% yield.December 19, 2011 at 6:05 pm #18143
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