biznatch's Content

#135497 Can I go for ChP having frozen tissue

Posted by biznatch on 05 June 2012 - 03:40 PM in ChIP and Next Generation Sequencing

We do ChIP from frozen samples all the time. The tissue is allowed to thaw slightly on ice, minced on ice with sharp dissecting scissors, resuspended in DMEM (no FBS) then passed (scraped) through a nylon cell strainer into a 50 mL tube with ~10 mL cold DMEM, everything is kept on ice. Once the single cell suspension in DMEM is prepared formaldehyde is added to 1% final concentration and the protocol proceeds as normal.

#135487 Which beads should I use with IgM antibodies for ChIP?

Posted by biznatch on 05 June 2012 - 11:20 AM in DNA Methylation and Epigenetics

I want to do ChIP for Polymerase II phosphoserine 2 and phosphoserine 5 in mouse tissue. I can use these antibodies, which are mouse IgM antibodies. Should I use protein L magnetic beads or anti-mouse IgM magnetic beads to bind the antibodies after IP? I found this document which says that protein L should be used for mouse IgM antibodies as long as "the immunoglobulin has the appropriate kappa light chains". How do I know if the PolII antibodies have the appropriate kappa light chains? I found one paper that used these specific PolII antibodies with these specific protein L magnetics beads, but other papers use anti-mouse IgM beads (not necessarily the ones I've listed, I would like magnetic beads though).

#135447 Experimental set up for qRT PCR

Posted by biznatch on 04 June 2012 - 01:28 PM in PCR, RT-PCR and Real-Time PCR

I think my link for the GenEx instructions didn't work.

www.bio-rad.com/LifeScience/jobs/2004/04-0684/Manual_Rev_B.pdf

These are the instructions for use the spreadsheet.

#135445 Experimental set up for qRT PCR

Posted by biznatch on 04 June 2012 - 01:04 PM in PCR, RT-PCR and Real-Time PCR

The Excel spreadsheet from BioRad that I linked to will let you input ct values and will normalize to your housekeeping gene and output fold change values.

#135220 Experimental set up for qRT PCR

Posted by biznatch on 30 May 2012 - 09:20 AM in PCR, RT-PCR and Real-Time PCR

I think you're using too much starting material in your qPCR reaction which is why the Gapdh amplification curve looks strange. You generally don't need to degrade the RNA after making cDNA. I would do a 10-fold dilution series (10, 100, 1000, 10000 fold dilutions) and see what your amplification curves look like.

#135186 Using 4% PFA in the lab

Posted by biznatch on 29 May 2012 - 10:48 AM in General Lab Techniques

I don't do this in a hood but I'm usually only doing a few plates at a time and my the PFA is only open to the air for a few seconds. After I wash with PBS I quickly unscrew the lid of my PFA tube, add some to my plates of cells, put the lids back on the plates and recap the tube of PFA. When they're done fixing I remove the lid from the plates and immediately asperate the PFA then continue PBS washing.

#135182 Experimental set up for qRT PCR

Posted by biznatch on 29 May 2012 - 09:53 AM in PCR, RT-PCR and Real-Time PCR

You may be starting with too much template for such a highly expressed gene. A dilution series (standard curve) with your Gapdh primers will tell you if/how much you need to dilute your starting DNA when measuring Gapdh. Do you know approximately how much cDNA you're starting with? Eg. I start with 0.5-1 ug of RNA, make 50 uL cDNA, then use 0.4 uL cDNA in each 20 uL qPCR reaction. This works well for both high and low expressing genes. In regards to how to set up your plates, if for example you have 3 biological replicates (6 samples altogether: 3 normal and 3 stressed), and wanted 3 technical replicates for each sample, I would set it up like this:

Posted Image

If you only have one biological replicate for now that's fine you can do the other ones later. The only thing you have to repeat every time is the no template control ("blanks").

We use an Excel spreadsheet from Biorad for our ct/expression analysis, though there are probably better/easier ways to do this. It's not the greatest (eg. your data has to be layed out perfectly before importing it) but we've been using it for years and once you get the hang of it, it's easy. You just need to input the ct values so you can take data generated at any time from any machine. Most qPCR machines should be able to output an excel sheet or txt file with the ct values (we use a Opticon Monitor from Biorad, also an old program). It basically automates your calculations, including normalizing to a housekeeping gene and calculating fold change. If you leave efficiencies at 100% you're using the ddct method, if you calculate efficiencies using a standard curve and give that data to the spreadsheet you're using the Pfaffl method (information on the two methods, at the bottom of this page).

Here's the spreadsheet:
www.bio-rad.com/LifeScience/jobs/2004/04-0684/genex.xls

and here's some instructions:
www.bio-rad.com/LifeScience/jobs/2004/04.../Manual_Rev_B.pdf

This spreadsheet shows on the left how I would organize my data before importing it into the spreadsheet from Biorad, and on the right where each value would end up once imported:
https://dl.dropbox.c...me%20setup.xlsx

Everything in the first three columns in red would be copied then imported in to the Biorad spreadsheet. Note how each is labelled and how the coordinates are organized.

#135169 How & where to prepare qPCR

Posted by biznatch on 29 May 2012 - 06:21 AM in PCR, RT-PCR and Real-Time PCR

My lab does tons of PCR and qPCR. Everyone sets their PCR up at their own lab bench that they use for all their other work. No special cleaning, no special pipettes, no problems with contamination. We use filter tips for anything PCR related, and use Milli-Q water. I aliquot my Milli-Q water in 1.5 mL tubes so that any given tube of water is only used for a few PCR's. If I use the water from say a 50 mL tube, I usually start seeing contamination after 10 or 20 PCR's.

#135168 data analysis for RT_PCR

Posted by biznatch on 29 May 2012 - 06:14 AM in PCR, RT-PCR and Real-Time PCR

Expression of your housekeeping gene can change over time. I would use at least two independent housekeeping genes in a timecourse experiment (eg. Gapdh and Bactin), at least until you establish that your housekeeping gene is consistent.

#135152 Second round of PCR after gel extraction fails miserably

Posted by biznatch on 28 May 2012 - 10:15 PM in PCR, RT-PCR and Real-Time PCR

I've successfully used gel extracted PCR products as template for a second round of PCR, though nothing as big as 3-5 kb, and only once or twice. It looks like you're doing about 30 cycles in your first PCR? What about just increasing to 35 or 40 cycles, maybe then you won't even need the secondary PCR?

Otherwise nested primers sound like a good idea. Without nested primers, the primers for the second round of PCR have to bind right at the very end of the template. Maybe a bit of degradation knocks a base pair or two off the end making binding much less efficient (I'm just guessing here). I know that restriction enzymes don't bind efficiently right at the end of a DNA strand, maybe primers don't like to either (more guessing Posted Image

).

I read in a forum a while ago that someone would take a needle and poke it into the gel into the PCR product band, then just stick the needle tip into the PCR mix for the second reaction. Enough DNA would be transferred just on the needle tip to reamplify it. I've never tried this so I can't comment on how well it works.

#135151 Amplify cDNA for qRT-PCR?

Posted by biznatch on 28 May 2012 - 10:01 PM in PCR, RT-PCR and Real-Time PCR

By pre-amplification kit do you mean a reverse transcription kit, ie. a kit to made cDNA from RNA? We've been using the Qiagen SuperScript II Reverse Transcriptase kits for years before doing real-time PCR. We usually start with 0.5-1 ug RNA in a 50 uL reaction (scaled up from the kits 20 uL specification) but use half the recommended concentration of enzyme since it's kinda expensive, and we use a lot.

#135150 Experimental set up for qRT PCR

Posted by biznatch on 28 May 2012 - 09:51 PM in PCR, RT-PCR and Real-Time PCR

I will do that in same plate, but annealing temperature for each gene is different. Therefore I can't run them on same time.

You don't have to run all your genes at the same time. You don't even have to run them on the same day. As long as you're consistent and proficient with your PCR set up you'll get the same results whether you run them together or seperately. You should however keep your biologcal samples together ie. if you're comparing expression of a given gene in one normal vs one stress condition, run both that normal and stress condition for that gene in the same run. If you're not sure just how proficient you are, then repeat some of your PCR after a few days and see if it's consistent.

#135149 What went wrong in my qPCR?

Posted by biznatch on 28 May 2012 - 09:30 PM in PCR, RT-PCR and Real-Time PCR

Hi,

I am new in qRT PCR. I ran around 5 times to troubleshoot my qRT PCR result. In mixture I am using double distilled autoclaved water and using SYBR green for this.

In disassociation curve I am getting multiple peaks. I know it can be because of primer dimer. But I am getting desired length of product which is required when I ran sample on gel after qRT-PCR. It just one band I am able to see when running qRT PCR product on gel. I am using 50Microlitre of reaction mixture for one well. I am using dliuted SYBR Green (10,000X) to 1X, .01X, .05X, 0.5X. I thought might be SYBR green is an issue, therefore I diluted it more.

I am unable to figure it out, can you please help me.

I've had situations like this. Sometimes you'll see just one band on the gel but get multiple peaks in the melting curve. This could be because there are actually two peaks on the gel but they are too close together (running your gel longer may help) or there could be faint secondary products that you can't see on the gel, as the realtime PCR melt curve is a lot more sensitive that an agarose gel. Unless both my gel and melt curve indicate one clear product I don't trust the results and I either optimize the primers or design new ones. You could try an annealing temperature gradient and see how that affects what the melt curve and gel look like.

#135148 A faster way to pick single colony clones for PCR screening?

Posted by biznatch on 28 May 2012 - 09:10 PM in Molecular Biology

I've done something similiar to the above post.

I set up a gridded plate and a complete PCR mix in uncapped PCR tubes (strips or plates). I use a sterilized pipette tip just held in my clean gloved hand to scrape off the colony, scrape a bit onto the gridded plate, then place in a PCR tube and let it sit there until I've collected all the colonies. An 8x8 gridded plate works well with 8-tube PCR strips. It's very fast, and you'll get enough cells on both the gridded plate for more bacteria to grow and in the PCR mix for a strong PCR reaction.

I originally would just put in in water then add a few uL of the water to the PCR mix, which worked, then to go faster I tried with a PCR master mix minus the Taq (didn't want Taq sitting in the MM for so long) and that worked, so I went ahead with the Taq pre-added and it also worked. I'm not even using hot-start Taq. If I have more than one plate to do I'll split it up so the MM + Taq doesn't sit at RT for more than a few minutes. ie. do one plate's worth of reactions in one PCR block and start it, then set up a new MM for each subsequent plate.

#135144 PCR product one day, none the next day

Posted by biznatch on 28 May 2012 - 06:16 PM in PCR, RT-PCR and Real-Time PCR

Very late followup, but if I want to thaw something quickly I put it in room temperature water. Usually it's just 1.5 mL tubes so I have the bottom half of a P1000 pipette tip box on my desk with one of these sitting in it (it just fits) and it's filled with water. Water transfers heat way faster than air so tubes will thaw much faster but won't get any warmer than ambient.

#135141 How should I optimize my PCR

Posted by biznatch on 28 May 2012 - 06:01 PM in PCR, RT-PCR and Real-Time PCR

Based on your really, really bright bands at the correct size, I would use less starting DNA or fewer cycles.

What are your cycling parameters? With a 284 bp amplicon you can extend for 20-30 seconds even with low quality Taq, less for better Taq. This may help limit formation of the bigger, non-specific bands.

#135140 Real time data analysis using 2 machines

Posted by biznatch on 28 May 2012 - 05:51 PM in PCR, RT-PCR and Real-Time PCR

It depends what's done on each machine. For example let's say you have two genes (X and Y) and two treatments (C and KO) and you want to know how the expression of each gene is changed in C vs KO. I would make sure that both C and KO from gene X are always run together (same machine and at the same time), but you could run C and KO from gene Y on a different machine or on the same machine but at a different time. If gene X is increased 3 fold in KO over C, it shouldn't really matter what machine you use, it should always be 3 fold increased. Though as with anything there may be slight variations. I definately wouldn't run C on one machine and KO on the other and try to compare them.

#135138 chip seq protocol

Posted by biznatch on 28 May 2012 - 05:17 PM in DNA Methylation and Epigenetics

Farnham lysis buffer uses NP40 as a detergent to lyse the cell membrane but it not the nuclear membrane. The nuclei are then subsequently transferred to an SDS nuclear lysis/sonication buffer to lyse the nuclei and release the chromatin.

If you use an SDS lysis buffer from the start you can perform all the lysis and sonication in the same buffer, but you may get interference or background from cytoplasmic proteins.

Here's some good information:
http://www.abcam.com...ource&rid=11385
http://www.protocol-...osts/33794.html

#135137 Bisulfite cloning... reading material

Posted by biznatch on 28 May 2012 - 04:49 PM in DNA Methylation and Epigenetics

Your product will have millions of copies of your DNA of interest, but each copy represents one allele from one cell of the starting material (eg. if you had 100 cells there would be 200 possible alleles). If you sequence it directly you'll get a mixture T's and C's in your sequencing results at each CpG site, since some alleles are methylated and some are unmethylated, and you won't be able to differentiated between a methylated or unmethylated state at any given site (unless it's 100% unmethylated or 100% methylated). When you clone the PCR product, a single piece of DNA gets cloned into each bacteria cell so each colony will represent a single allele from your starting material. You then have to PCR amplify from each colony to get enough DNA for sequencing, but in this "colony PCR", you will end up with millions of copies of that one allele so it will be "pure" for either C or T at each CpG. If you colony PCR amplify and sequence 20 colonies then you're generally looking at 20 representative alleles likely from 20 different cells, and you can see what proportion are methylated and what proportion are unmethylated. Note that sometimes you can get the same allele in more than one colony since your original PCR step generated many copies of each allele. If you sequence 20 colonies it might actually represent 18 alleles plus two PCR duplicates. To differentiated between alleles you should look for variation in CpG methylation and conversion at each C, including non-CpG C's. It is unlikley that two different alleles will have the exact same patterns for both of these things.

I hope I explained that ok, it's kinda confusing. I don't know any specific reading material I just learned bisulfite sequencing from others in my lab and the lab next to us. Feel free to ask for any clarifications.

#135136 Problem of Direct bisulfite sequencing

Posted by biznatch on 28 May 2012 - 04:38 PM in DNA Methylation and Epigenetics

Maybe I'm missing something, but aren't you supposed to clone the PCR product before sequencing? When I did this I gel purified, cloned, then sequenced a bunch of clones.

#135134 What 260/280 and 260/230 ratio should I expect from sonicated chromatin?

Posted by biznatch on 28 May 2012 - 03:22 PM in DNA Methylation and Epigenetics

When I use a Nanodrop to check PFA fixed, sonicated chromatin my 260/280 ratios are 1.2-1.7 and 260/230 are 0.4-1.4. I don't know why the 230 ratio is so variable. It varies from one prep to the next but is usually consistent within the prep; one time all the samples will be around 0.4-0.5, another time they will all be around 1.2-1.4, though the samples are prepared the same way. I'm trying to troubleshoot ChIP and am wondering what I should expect. I think the ratios should be lower than pure DNA because there is protein there as well but I don't know what is a good range for the two ratios.

#118530 PCR product one day, none the next day

Posted by biznatch on 30 August 2011 - 07:30 AM in PCR, RT-PCR and Real-Time PCR

I've had some cDNA that worked for some genes and not others, even though with other batches of cDNA all the genes work. I'm not really sure why this is the case. It kinda seems like with primers that work really well and genes that are highly expressed, they will PCR amplify even with poorer quality cDNA. Can you try designing different (better?) primers for the gene that's not working?

In regards to storing cDNA, it's more hardy than you think. I've never heard of storing it at -80. I store my cDNA at -20 and freeze/thaw it many times without any degradation. The problem I describe above occurs from immediately after the cDNA is made, it's not a freeze/thaw issue. I've forgotten cDNA in the fridge for several days, and at room temperature overnight, without any problems (although I wouldn't recommend that!).

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