49 replies to this topic

[epimaster]

Hi Guys, I wanna do long PCR around 2Kb PCR product but I dont know which polymerase I can use, I know Pfu works well but there are some other options? Thank you for any reply  

[epimaster]

Hi Guys, I wanna do long PCR around 2Kb PCR product but I dont know which polymerase I can use, I know Pfu works well but there are some other options? Thank you for any reply

[Adrian K]

try using phusion flash polymerase or KOD polymerase...

[beta]

Hi,
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).

Any suggestion/recommendation?

[phage434]

Suggestions:
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.

[boaziga]

pcrman, on 01 April 2004 - 09:57 AM, said:

I've used qiagen kits a lot and never encountered similiar situation.

Quote

QIAQuick PCR purification kit: but when I run the gel to see its efficacy, there was simply nothing!! it cleaned everything including my band!!

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.

[Sunflower_gl]

Boaziga: How can you estimate the electrophoresis time and the voltage necessary for having the best result? I put my PCR product on the gel about 1h at 150V, but i have no band.
Thanks in advance

[Trof]

The voltage should be proportional the distance of the electrode, maximum around 7V/cm of electrode distance. The time depends on the migration of the loading dye, on 1% gel the bromphenol blue travels around 400 bp. If you use in-gel ethidium bromide, you can put it out of the tank and check under UV, if you're not sure.

When you say you have no band, do you mean you don't even see the marker on the gel?

[Sunflower_gl]

Trof: Thanks for your advices. The marker was seen clearly on gel of course, but i don't have any band of my PCR product. I will verify the electrode distance in my labo.

[Trof]

If you got nice marker bands and no PCR product, then the problem most likely is not in the electrophoresis, but in the PCR.
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.

[Sunflower_gl]

You're right. I've just tested another RT-PCR, i have nice marker bands but no bands of my PCR product. Here is my PCR protocol and its cycle profiles:
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.

Cycle profiles:
Hot Start: 91°C for 3 min
Cycles- 34
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?

[leelee]

The product manual says to use a hot start temperature of 95C for 15 minutes- so looking at your cycle conditions, I'd hazard a guess to say this is where your problem is.

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.

[Sunflower_gl]

Thanks all of you guys. I've changed my PCR program as the advices of Leelee and i've got nice bands of my PCR products with CD45 and FLNA. That's great for beginning others experiences!
Have a nice week!

Sunflower_gl

[Sunflower_gl]

Hi guys,

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!

Best
Sunflower-gl

[Sunflower_gl]

Hi there,
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?
Thanks