http://www.protocol-online.org/forums/index.php Fri, 20 Nov 2009 18:23:58 -0800 60 http://www.protocol-online.org/forums/index.php?showtopic=11695
Hello!

I was wondering how to get labelled nucleotides, if you can buy them or make them urself (if so where can I find protocols). I know you can radioactively label them, theres also biotin....but ive been looking for a protocol or something and I can only find papers where people use them. I also looked for radioactivaly labeled SAM and was unsuccesfull.


Would neone know of a protocol or company and maybe pros and cons of using labeling?


Wish you all a great weekend!]]> Fri, 20 Nov 2009 06:27:45 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11695 http://www.protocol-online.org/forums/index.php?showtopic=11691
I'm really unhappy with my DNA isolation at this moment since I cannot get ride of RNA from the sample, which I've heard will interfere with DNA during PCR. For removal of RNA I did RNAse treatment and then phenol extraction and CIA (chloroform+isoamyl alcohol) extraction. The DNA was precipitated using NaOAC and absolute ethanol, pelleted, washed with 70% ice-cold ethanol, dissolved in TE solution and the concentration was measured using Nanodrop. The ration 260:280 is all the time around 2. I repeated RNase treatment in same sample but no improve.
To look for the presence of other stuffs along with DNA, I also did ran agarose gel with the sample. Here is also the photo of the gel (sample and ladder). It seems from gel my DNA is not pure. I require some help, please!
http://www.protocol-online.org/forums/index.php?act=attach&type=post&id=970]]> Fri, 20 Nov 2009 03:38:37 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11691 http://www.protocol-online.org/forums/index.php?showtopic=11690 For some time now we polyadenylate in vitro transcripted and purified mRNAs with polyA polymerase from USB, but all of a sudden it stopped working.
We tried different polymerases, different templates and different ATPs. We also tried different purification methods before polyadenylation (3 Kits, LiCl and nothing at all).
Does anyone have an idea what else we could change so it works again?
Thank you soooo much.
Schnuffel]]> Fri, 20 Nov 2009 03:04:08 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11690 http://www.protocol-online.org/forums/index.php?showtopic=11672
I need to isolate specific fragments from a RACE (Rapid Amplification of cDNA Ends) reaction. Classic RACE reactions work similarly to PCR reactions, the drawback is the great amount of aspecific products that usually appear as a smear when run in an agarose gel. I would like to know if anybody knows a method to specifically precipitate (through hybridization) the specific products (alternatively I could extract them from an agarose gel, if recognizable). I would like to proceed so:
1 RACE reaction
2 hybridization with a specific DNA oligonucleotide( I already have it because of other purposes)
3 product identification and isolation

Does anybody have any idea (or protocol)?]]> Thu, 19 Nov 2009 08:53:35 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11672 http://www.protocol-online.org/forums/index.php?showtopic=11670 my friend with the same master mix volume, but different primer was able to get the band, is that the primer problem?
does primer expired?]]> Thu, 19 Nov 2009 04:00:33 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11670 http://www.protocol-online.org/forums/index.php?showtopic=11662 ]]> Wed, 18 Nov 2009 23:04:47 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11662 http://www.protocol-online.org/forums/index.php?showtopic=11661 I am trying to find out the molar concnetration of Guanidinium Thiocyante in Ambions Tri Regant . Is it 2M or 4M ? Wed, 18 Nov 2009 19:00:37 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11661 http://www.protocol-online.org/forums/index.php?showtopic=11660 In my experiment I get alot of colonies on my plate, but most of them does not contain my insert. I dephosphorylate my vector when i do my digestion, then i gel purified my vector and my insert. I nanodrop my vector and insert before i do my ligation. My vector concentration is normally 30ng/ul and my insert is 65ng/ul. I did a 1:2 and 1:3 ligation and O/N at 4 degree. Next day, i added 15uL ligation reaction into 50uL of my competent cells. Would this 15uL affect the up take of the right plasmids???

Another question is, in a 1:2 ratio ligation, I added 2uL of vector and 2uL of insert, is this a 1:2 ligation ratio in a 20uL ligation reaction??? and is adding 2uL of vector and 3uL insert a 1:3 ratio ligation???

Thank you

Jason]]> Wed, 18 Nov 2009 19:00:14 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11660 http://www.protocol-online.org/forums/index.php?showtopic=11656
We were given a question on an exam where we were to calculate the amount of enzyme needed for a 20 uL digest. I took into account the reduction of volume and had points taken off because my professor said that the new volume did not have to be taken into account. My question is when does the final volume of the digest become a factor in how many units of enzyme you add? Using my professor's answer, I could perform that digestion in a bath tub as long as I keep the amount of enzyme and DNA the same which cannot be true. Any help would be greatly appreciated! ]]> Wed, 18 Nov 2009 15:36:13 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11656 http://www.protocol-online.org/forums/index.php?showtopic=11653
So, im thinking of purifying the labeled probe from a gel to isolate the right band.. i'm worried the EtBr in the gel may interfere with DNA binding, is that a valid concern? ]]> Wed, 18 Nov 2009 13:44:31 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11653 http://www.protocol-online.org/forums/index.php?showtopic=11650 thanks in advance]]> Wed, 18 Nov 2009 12:01:12 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11650 http://www.protocol-online.org/forums/index.php?showtopic=11646
I have paraffin tissue sections on glass slides that I need to microdissect. At the moment the tissue is really stuck on the slide and even if I cut around the area of my interest, it is hard to get the tissue off.

I am wondering if you guys know a method which allows me to lift off the sections and place it onto membrane coated slides. Any suggestion (chemical, thermal..) would help.
Cheers,

Richard]]> Wed, 18 Nov 2009 07:40:32 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11646 http://www.protocol-online.org/forums/index.php?showtopic=11644 I tried to extract Fosmid dna using the Epicentre FosmidMax purification kit. When I ran the gel there was no band. But when i checked the concentration in spectrophotometer it showed a concentration of 550 ng per microliter. what cud be the reason. can nyone help. Wed, 18 Nov 2009 07:05:58 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11644 http://www.protocol-online.org/forums/index.php?showtopic=11635
Pls help asap
ASM]]> Wed, 18 Nov 2009 02:30:29 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11635 http://www.protocol-online.org/forums/index.php?showtopic=11620
This Molecular Cloning Guides covers:

Subcloning:
Subcloning is a technique used to move a particular gene of interest from a parent vector to a destination vector in order to further study its functionality.

PCR Cloning:
Use PCR reactions to amplify a insert DNA fragment, followed by sub-cloning (restriction endonuclease digestion of insert and vector DNA and fragment ligation).

Gene Synthesis:
Use DNA oligos and PCR reactions to synthesize a gene without source DNA as template followed by Subcloning (restriction endonuclease digestion and fragment ligation).

TA TOPO Cloning:
TA Cloning is a subcloning technique that doesn't use restriction enzymes and is easier and quicker than traditional subcloning.

Directional TOPO Cloning:
Use PCR reaction to amplify a DNA fragment. The resulting PCR products have four additional bases (CACC) at the 5´ ends that are from the specially designed forward PCR primer. With a special ligation kit, this fragment is directly ligated into a linearized vector DNA (D-TOPO Vector, which contains GTGG overhangs at the 5’ end) without pre-digestion with restriction endonucleases. The fragment can only be inserted in forward orientation.
Gateway Cloning:
This is a cloning method based on the site specific recombination of lambda bacteriophage.

]]> Tue, 17 Nov 2009 14:31:42 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11620 http://www.protocol-online.org/forums/index.php?showtopic=11610
I'm starting a new lab and been having problems with a western blot. I have tryed everything and I have no signal!

People here told me once the lab fridge went out of current a whole weekend and all antibodies are there! My boss says nothing happened, that abs can resit that, but now I'm not so sure (luminol was there too by the way).

I did dot blot just with my secondary on PVDF membrain, (1:10 and 1:10 000) and saw nothing =(

What do you think about it?]]> Tue, 17 Nov 2009 09:52:38 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11610 http://www.protocol-online.org/forums/index.php?showtopic=11607
The protocol of Qiagen's miRNeasy kit recommends the use of chloroform without added isoamyl alcohol. Can anyone reccomend which type of chloroform I can use? Amylenes and ethanol is added for stabilising the solution but can 0.5-1% harm the protocol?

Cheers]]> Tue, 17 Nov 2009 07:23:18 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11607 http://www.protocol-online.org/forums/index.php?showtopic=11596 I need a vector that does not have ampicillin as resistance marker. Kan or tet or chlor is fine but no amp
Thanks]]> Mon, 16 Nov 2009 20:33:37 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11596 http://www.protocol-online.org/forums/index.php?showtopic=11590
1) I isolated mouse genomic DNA using Qiagen blood mini kit. The kit says that it can purify genomic dna up to 50kb, but predominantly its 20-30kb fragment. I did run a gel for the genomic dna isolated and they show a strong band around 20-30kb, with a little of smear above and below the band. I thought that as long as there is some genomic DNA larger than 28kb, it shouldn't affect my long pcr right? But just in case, i was thinking to isolate larger genomic DNA without buying another kit. I saw a standard protocol at this link, http://www.genome.ou.edu/protocol_book/pro...tIII.html#III.H. Does anyone know if this protocol allows high molecular weight genomic DNA isolation? Or does anyone with prior experience of cloning such large fragment knows if the genomic dna cloned via the qiagen blood kit is good enough?


2) Regarding the long PCR, i'm using Epicentre master amp extra long pcr kit. The cycling conditions are as follows: initial 94deg 1 min denaturation, 15 cycles 94deg 10 sec denaturation, 62deg 1mins annealing, 68deg 28mins extension, 15 cycles of the same thing but with extension temp increased by 10sec every cycle.

The annealing temperature of the primers are 66deg. I'm using 150ng genomic DNA (they recommend 100-500ng for 50ul reaction), 0.2uM primer each (final conc) for a total of 25ul reaction.

So my first attempt failed. I suspected its because of my primers, which was supposed to be 25bp and 24bp long (blasted and checked for dimers and other secondard structures). I ordered the primers with additonal 12bp at each end to include restriction site and GC over hang, thinking that it should be all right since we do that often for typical PCR.
Does anyone know if that could be the problem? Anyhow, i juz ordered the primers again, this time without any additonal sequence. I'm intending to retry the pcr again but i would like see if anyone find any problem with my pcr conditions or cycling parameters, or have any advice on it.

I'm thinking for the next pcr to perform a step down, to start with annealing temp of 70deg, decrease 2 deg every 2 cycles till 60degs. That is in addition to the cycling parameters i'm already using. Also, i'm going to include a final 10mins extention at 68deg.


Any other suggestions will be much appreciated. Sorry for the long question. Thanks]]> Mon, 16 Nov 2009 18:16:55 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11590 http://www.protocol-online.org/forums/index.php?showtopic=11589 I'm cloning my insert into pET15b vector. I did a ligation and transform them into DH5alpha. There are lots of colonies grew on the plate, then i screen them with colony PCR using the T7 promoter and terminator primers, as well as with my insert primer. Apparently, the PCR with my insert primers showed me products, but none of the T7 primers showed me products. So i decided to extract the plasmid from the colony that gave me positive result in my colony PCR with insert primer. Then I did a PCR using the T7 and insert primers again with the plasmid I've extracted. There were no positive results for my insert primers, but they were positive result for my insert primers. What is happening here?? I should able to get positive results with T7 primers because im cloning into pET15b. If my insert is not cloned into pET15b, where has it cloned into???

PLEASE HELP ME!!!!!

Thanks...]]> Mon, 16 Nov 2009 17:26:49 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11589