PCR product purification
Posted 01 August 2011 - 07:45 AM
Posted 01 August 2011 - 07:46 AM
Posted 01 August 2011 - 10:32 AM
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
Posted 23 August 2011 - 10:56 PM
I purify my PCR product using QIAGen Gel ext kit and i have no problem recover my dna however the purity of the dna recovered is less and 1.5 (A260/A280). Out of 10 replicate I made i only be able to obtain 2 replicate with desirable purity (1.8 - 2.0). I need to purity to be around 1.8-2.0 as it is the requirement for sequencing (outsource).
Posted 24 August 2011 - 04:44 AM
When doing your gel extraction, wash the column with pure buffer QG following the initial flow through.
Wash the column twice with buffer PE, making sure you flood the lip of the column with PE to clean it.
After emptying the waste, make very sure you are spinning the column dry at high speed. About 50 ul of liquid should come out of the column at this point.
Elute twice, first with 25 ul and a second time with another 25 ul. Deposit the elution buffer directly onto the bottom of the column with a tip. Waiting a minute or two after adding the elution buffer before spinning it down helps.
Posted 30 October 2011 - 10:11 AM
Did you run a gel before purification to make sure your PCR amplification was successful? I usually have two type of kits at hand, one is the PCR purification kit, the other is Gel purification kit. After each PCR, I run 4 ul of product to see what my amplification looks like. If there are strong primer dimers or non specific bands, I will run the whole PCR reaction in another gel and cut the bands followed by purification using the Gel kit; if the amplification is clean (desired bands are strong, no primer dimers or non-specific bands), I will go for PCR purification kit directly w/o further gel purification.
Hope it helps.
You may want to adjust your voltage and electrophoresis time. Usually bands over the gel and leave little or nothing if the time is longer than necessary and if the voltage is too high.
Posted 04 January 2012 - 06:48 AM
Thanks in advance
Posted 06 January 2012 - 01:48 PM
When you say you have no band, do you mean you don't even see the marker on the gel?
I never trust anything that can't be doubted.
Posted 09 January 2012 - 01:28 AM
Posted 11 January 2012 - 07:15 AM
If you seen "smiling" or other disruption of marker bands, or they didn't get separated right, then you should consider a problem in the ELFO.
I never trust anything that can't be doubted.
Posted 11 January 2012 - 08:19 AM
Buffer 10X 2,5µl; 2µl dNTP 10mM of each; 2,5µl primer fwd; 2,5µl primer rev; 0,5µl HotStarTaq (Qiagen, 2,5U/reaction); 2,0µl MgCl2; 2,5µl cDNA; 11,25µl PCR grade H2O for 25µl in total.
Hot Start: 91°C for 3 min
Denature: 91°C for 30 sec
Annael: 58°C for 45 sec
Extension: 68°C for 45 sec
Final extension : 68°C for 10 min
My RNA are extracted from human blood and the primer pairs that i used are CD45 and FLNA.
Do you have any experiences with HotStarTaq?
Posted 11 January 2012 - 08:45 PM
First thing I would try running the PCR exactly as the manual suggests.
When using a new polymerase, you should always read the product insert, as seemingly small differences in temperature can turn out to be crucial for reaction success.
Edited by leelee, 11 January 2012 - 08:46 PM.
Posted 16 January 2012 - 07:49 AM
Have a nice week!
Posted 21 February 2012 - 02:40 AM
I've done RT-PCR with some modifications like your suggestions and i had very nice bands with CD45 and FLNA primer for RNA extracted from human blood. But in my research i have to work with RNA extracted from human sperm. These RNAs are small fragment of 1000-2000nt. I've done the same RT-PCR program with RNAs spermatozoa samples but it didn't work well. Normally the band of 848pb must be appeared by migration on agarose gel electrophoresis (for CD45) but i have some "smear" bands about 200pb.
Have anyone got the same problem? Do you think i'd better change another kit RT-PCR? Actually i am working with Omniscript (Qiagen) and HotStarTaq (Quiagen) and i suppose that these reagents are not "sensitive" enough for amplifying small fragment like RNAs spermatozoa.
Any advices will be appreciated!
Posted 03 May 2012 - 05:35 AM
I can't figure out my problem with my PCR. I've tried to change Mg2+ concentration, annealing temperature... but nothing happen. I have always smear bands. my lab-mate (who has done many PCR) suggested me use the Q-solution to increase PCR specificity. But i have no experience with this solution. In fact, I am doing PCR with 4mM Mg2+ concentration and i don't know if I have to decrease the concentration of Mg2+ when I add Q-solution. Do you guys have any advices?