DNA Methylation and Epigenetics Forum

CHIP / Is it normal to see pellet after the DNA purification?

Mon, 21 Jul 2008 05:10:43 -0700

Hello

I m doing CHIP assays and during DNA purification and after treatment with 100 % and 70% ethanol and centrifugation I see pellet in all my samples. Is this normal to see the pellet-DNA? I am thinking that maybe I should reduce the amount of chromatin that I incubate with the antibody.

Thanks in advance

Amplicon size for BSP

Mon, 01 Sep 2008 02:11:04 -0700

Hi!!

Does anyone know the optimal amplicon size when you perform a bisulphite sequencing??

Thanx!!

problem with WGA amplification of my chip sample

Tue, 02 Sep 2008 14:08:44 -0700

Hi, I've been trying to amplify my chip samples with WGA kit from sigma. So far the positive control works perfectly. I get about 10ug from 10ng genomic DNA included in the kit. However, with same amount of chip samples I've only get 0.3-0.5ug. I searched the forum and it seemed that nobody has this problem before. It's so frustrating. Sigma technical support suggests that I can amplify for 20 cycles instead of 14 and they think iincreasing the cycle numbers to 20 will not introduce much more bias compared to 14 cycles. I'm wondering whether anybody has a similar experience before?

Thanks a lot. I appreciate your help.

Chip Troubles

Mon, 01 Sep 2008 21:09:34 -0700

Hello. I'm hoping someone might help me troubleshoot my ChIP experiment.

I'm trying to perform the following ChIP protocol for a transcription factor using a cell line that over-expresses a flag-tagged protein. My ultimate goal is to ChIP for endogenous protein in primary cells after getting the conditions optimized using my cell line.

In brief, I fix ~20x106 cells for 15' w/ 1% formaldhyde at RT, quench and then wash with PBS. I lyse the cells, spin, and then resuspend the nuclei in low salt (200mM) nuclear extract buffer followed by equal volume of a high salt (600mM) NE buffer.

I have no sonication problem as I routine obtain fragment sizes b/t 400 - 1000bp. Since many people seem to struggle with the sonication step, I sonicate my chromatin extract for a total of 60 seconds using a digital Branson sonifier at 60% power in 200uL volume. 1" on/ 1" off x10 pulses, wait 2' on ice and repeat 6 times. The small volume size (200uL) is crucial.

I use 25uL of Invitrogen's M280 strepavidin magnetic beads per IP. I first block the beads with salmon sperm DNA and then preincubate with a biotin anti-rabbit IgG for 4 hours in BSA/PBS, wash w/ BSA/PBS 3x, and incubate with either a rabbit anti-FLAG antibody, rab anti-TF antibody or pre-immune rabbit sera (no antibody control) overnight. Next, I wash and then incubate overnight with my chromatin extract from above at a final concentration of 20mMHepes, 0.5%TritonX, 150mMNaCl, +inhibitors.

The following day I wash the M280 beads with a buffer containing 500mM LiCl2 and 1% TritonX100 6 times for 5' each, followed by TE+ 50mM NaCl 1 time. I then elute with SDS/EDTA for 15' at 65C and reverse cross-links overnight. I phenol/chloroform, EtOH ppt. and perform real-time PCR.

I have the benefit of a positive control gene; someone has demonstrate binding of the TF to a particular gene.

My problem is that I obtain the same fold enrichment for my two negative control regions (one proximal to the positive control gene and another on a different chromosome - insulin promoter) as I do for my positive control region. The signals I obtain for the 'no antibody control' IPs are typically 4-fold weaker (dCp=2) than for the anti-FLAG and anti-TF IPs for each target region. This difference seems too small. The major problem is that both my anti-FLAG IP and anti-TF IP give very strong signals for my positive control region and, unfortunately, for my two negative control regions.

Here is a typical result:
Cp values

Specific Questions:

I pre-bind the antibodies to the M280 magnetic beads instead of first adding the antibody to the chromatin extract and then collecting with the beads. Should this matter? Has anyone compared the two methods?

I tried pre-clearing my chromatin extracts w/ M280 resin, but this resulted in a reduced fold difference for all my samples.

Has anyone used a more stringent washing regime than the 0.5% TritonX / 0.5M LiCl2-containing buffer?

It seems that any anti sera or preimmune sera I use will co-IP DNA. So, I next plan to spike the IP reactions with BSA to block nonspecific binding of my antibodies to the chromatin Is this a good or bad idea? If good, how much BSA should I use? Would it also be wise to spike the chromatin extract with salmon sperm DNA?

If anyone with experience troubleshooting this technique could give me some pointers as to which other steps of my protocol I should vary and test, I would be very appreciative.

Thank you for reading,

-ds

Methylation troubleshooting

Mon, 01 Sep 2008 07:15:03 -0700

Hi!

I am performing methylation assays, using the NEB enzymes (Msss I, and I also tested Hha I )
But I am experiencing problems of efficiency during the methylation step.

I proceed according to the protocols given with the enzymes (SAM conditions, DNA quantity, 1H at 37°C) and still, the methylation of the CpG islands doesnt seem to be optimal.
After the methylation I do a bisulfite treatment (that is working very well according to my controls)
Still, the sequencing of my genomic DNA shows a partial methylation. My concern is that I dont want to methylate too long (as the next step would be to proceed on nuclei and localize nucleosomes on my locus)

Similar experiments have been done using NEB enzymes, and it was shown that 15 minutes are sufficient while 1H is not enough in my hands!

So, I would like to know if there are any steps of the protocol that I should do with special care? Could the quality of the DNA interfere with the methylation? (anway, I purified my DNA and the ratios and salts are good)
I also tested diverse concentrations, volumes and quantities

Thank you very much for your help,

Marie.

DNA purification after sonication

Fri, 22 Aug 2008 02:12:14 -0700

HI everyone,
I am tring to establish ChIP in my lab, and now I have some problems.
After the sonication, I want to verify the size of fragment. Do anyone have a protocol for the DNA purification by chloroform/phenol after sonication? And do you reverse-crosslink before going into DNA purification?
Sorry for these questions which might be idiot for you. I really need some helps.
Thanks!

Success with Direct Sequening of Bisulfite Pcr Products

Fri, 24 Mar 2006 09:39:46 -0800

Hi All,

I've been working on developing a system to check the methylation status of 85 CpG islands across a 700 bp region (1152 bp is actually sequence used for calculation of primers). It has been a bit of a headache getting things to work right and in some cases I ended up just being lucky.

So here is what I wanted to share, good and bad, dumb and even dumber at times .

First attempted to design primers, using manual conversion of Sequence to reflect Bisulfite conversion. I then used primer3 and went from there. First plan was to do 3 pcr reactions of around 300bp to cover the 700 bp region with some degree of overlap for primer binding locations.
Spent a good month, with bad sequencing, boss not happy, bad pcr products .

Primer set A (1-355) Fair Product, Fair Seq
Primer set B (300-600) Good Product, Good seq
Primer set C (550-750) Fair Prodcut, No seq
Primer set D (600-850) Multiple Band Product No seq
Primer set E (625-875) Multiple Band Product No seq

One afternoon after reading this forum on Nested pcr I went back to my PCR primers and found a few primer pairs that would yield larger pcr Products 700+ bp

Primer set A 5' + Primer Set D 3'
Primer set B 5' + Primer Set C 3'
Primer set A 5' + Primer Set E 3'

I then checked on primers locking the 5' Primer and searching for a 3' primer. Tested all my current primers. Dumb luck kicked in

Primer set A 5' + Primer Set B 3' 450 bp
Primer set B 5' + Primer set C 3' 545 bp

So here is actually what I do.
Round 1
PCR Bisulfite treated DNA with Large primer set (A5+D3), (A5+E3) With pcr Tm 1 degree below calc Tm, ext temp 72 , 6 min final 72 extension , Sigma Jumpstart ReadyMix Taq, 2x 3' primer
Round 2
Using 2 ul of product from Round 1 , 2x 3' primer PCR with primer set (A5+B3, B5+C3), Tm 1 Degree below calc Tm , ext temp 68 , 6 min final 68 extension, same taq.

Gel extract , Purify , Spec and submit for sequencing.
Here is where this is a bit differant for sequencing,

I use the following primers for sequencing,
Product (A5+B3) sequencing primers (A3, B5)
Product (B5+C3) sequencing primers (B3, C5)

Visual example
Pcr Product (A5+B3)
[A5-------------->-----------------------------------------------------------------<----------------B3]
Seq primers underlined
[----------------------------------------------{B5------->--<----------A3}---------------------------]
90% of the time the 5' seq primer has enough coverage to reach the end of the product.

Depending on the primer sets I do however have around a 15bp gap I simply can't sequencing results in, no matter how hard I try or with which set I try.

While this method is somewhat sloppy, and I learned alot from my mistakes. I am now getting good data. Given the budget of my project, Time, and other factors, It worked for me and I might work for someone else. So I thought I would share this with all of you.
as an added bonus the product from round one can yield up to 25 pcr reactions for round 2 , minimizing my need to do bisulifite conversion.
Now I score the CpG methylation by measuring height peak setting 100% to be equal height peaks and score on a scale of 0-4 I try to average the back ground C signal and subtract from peak height for calculation. (Im still debating on if this is a correct method to use)

0 = No Methylation signal
1= <25%
2=25-50%
3=50-75%
4=>75%
666872 peak 44 = 4, 49 =3
666873 peak 48= 2 (barely)

Open for discussion , feed back or better hints.
I'm still refining the system to yield better results.
The future plans include 5-aza treatment (1.0 uM appears to yield 20% death at 48hrs with 2.5 uM killing around 80% at 48hrs ) trypan blue and MTS assay.

Best of luck
AD5

LONG AMPLICON PROBLEM

Thu, 28 Aug 2008 10:18:54 -0700

I'm having problems with primer efficiency. The primers I use amplify a product of 300pb. I have used the reaction protocol I have used before with 100pb product. I usually do 65ºC for 1 min for annealing and 95ºC for 1 min for melting. Should I add an extension step in the reaction? or maybe optimize dNTP concentration?

question about MSP bands

Wed, 30 Jul 2008 10:20:05 -0700

Hi!!

I'm doing MSP for different genes in different cell lines. In some cases, I observe a band when I use primers for unmethylated sequence and, in others, band when I use primers for methylated sequence. My question is... what happens when there are bands in both, primers for unmethylated and methylated sequence??? is the gene methylated or not??
Thank you for your help!!

A single band after sonication?

Wed, 27 Aug 2008 19:04:10 -0700

I got a problem with sonication in preparation of ChIP assay

I start out with some mouse liver frozen tissue

I thraw them on ice, cut them into small pieces by razor blade and homogenize them with a glass homogenizer in solution with sucrose and protease inhibitor (supposed to leave the nucleus intact)

Then I centrifuged the sample at 4C 2400g 10min to get a pellet, wash it with ice cold PBS and centrifuge again

The pellet is then resuspended in upstate ChIP assay kit SDS lysis buffer (100mg tissue -> 1.2ml buffer)

I split them into 300ul each and sonicate with 4 different conditions

mosonix 3000 with microtip, output 1.5, 10sec on, 20sec off, X4, X6, X8, X10 times

Then I did NOT decrosslink the DNA and went ahead to run a 1.5% agarose gel with 1X TAE

I couldn't see a clear smear whatsoever, and there is a quite discrete band at about 500-750bp

I tried to treat the sample with RNase A for 30mins and run the gel again, the band is still there

Did I do something wrong??

ChIP assay sonication problem

Thu, 12 Jan 2006 12:24:54 -0800

hi everyone:

I am having similar trouble with ChiP sonication step. I follow the protocol from Santa Cruz, and use the reagents as per manufacture protocol. However, when I sonicated my 293T cell nuclear extracts reversed cross-link and phenol extracted the DNA, I only got a smear of DNA.

I used the following conditions:

cross-linking: 1% formaldehyde - 10min, 37oC
quenching: 0.125M Glycine.

mosonix 3000 with microtip, 15sec, 12X, at 1.5 continous output setting.
Volume: 500ul.

reverse: 65oC water bath 4hrs.

phenol extract, then RNase Treat 37oC 30min (did not perform protein K digest as phenol removes most of the protein.)

I am not sure what I am doing wrong. any suggestions?

busy monkey

how to fix embryoid body (EB) by formaldehyde

Tue, 26 Aug 2008 07:27:52 -0700

As is known to all, formation of embryoid body (EB) is essential during in vitro differentiation of ES cells. I have to use such EB body to do ChIP assay. EB body is a clump of cells. I have no idea how to fix them. Should I disperse the EB body into single cell by trypsinization before fixation? Has anyone handled with EB body? Please give me some suggestions! Thank you, all guys!

CHIP SER 28 H3 PHOSPHO

Wed, 27 Aug 2008 10:45:20 -0700

HAVE TO DO SER28CHIP PHOSPHO.ANYBODY TRIED BEFORE THE ONE FROM UPSTATE?
DOES IT WORK?
THANKS

Primer dimer problem in MSP

Tue, 26 Aug 2008 01:05:41 -0700

With many help from here, I finally got specific MSP bands for several primer sets.

However, for one primer set, there still is a band below 100 bp, which I guess is a primer dimer.
(I ordered the primer that had been used in two papers)

There is also the specific right-sized band of ~200 bp. The purpose of this experiments is to quantify the methylation of the gene by MSP.

1. Can I just compare the amount of PCR band at the specific size even if there exists a primer dimer band? or should I find the PCR condition that brings no primer dimer band?

2. Then, how can I get rid of the primer dimer band? I am using hotstart Taq and primers at concentration of 200nM, and what do you recommend I should try next?

DNA methylation analysis - doubts

Thu, 21 Aug 2008 14:53:50 -0700

Hi everyone,

I am a new PhD student in this field and i am doing some bioinformatic work before DNA methylation analysis. However, my current supervisor is not of this field either, and the one that will help me with this will only come in October. So, as you imagine, i have a lot of questions...
Could you help me?!

My first task was to search for CpG islands in several genes, and I used MethylPrimer Express. I am now trying to get MSP (or BSP, i am not sure yet which one i will use) primers. This software provides me already these primers but I am not sure if i can use them directly or if it is better to check them with another software. Do you recommend one?
I found 3: PerlPrimer, BiSearch and MSPprimer but I don't know exactly how they work because i can't find good tutorials.

Also, after designing the primers, which are the best companies to produce them?

Another question: is there any BSP/MSP technique that would allow me to analyze several genes at the same time? (I need to study methylation of 10 genes in 4 different tissues of CTRL versus diseased mice of 3 different ages and this would take me too long to study one by one!!!)

Thank you very much
Cheers
Cris

Panomics Promoter Methylation PCR Kit and Methylation specific Digests

Tue, 05 Aug 2008 14:04:24 -0700

Hi,

I'm trying to determine the methylation status of my promoter of interest and would love some advice. I've tried doing bisulfite sequencing which was inconclusive, so am taking a step back to first determine if in fact my promoter is methylated. I wanted to ask if anyone has used the promoter methylation PCR kit from panomics and if they have had success.

I also want to simply run a digest (to determine if there is a genomic methylation difference between cell lines) with methylation specific or sensitive res. enzymes (which would be better?) on genomic DNA and was hoping someone could give advice on which enzymes to use and the order in which to use them (ie should I cut the genomic DNA isolated from the cell line (I have a cancer cell line and a wild type I will be using as a control) with an ezyme to make smaller fragments followed by methylation specific or sensitive enzymes?).

Any help would be appreciated,

Thanks,

Matt

Finding out what sequence to study by ChIP

Mon, 28 Jul 2008 11:12:58 -0700

Hi everone,

I am completely new to this field and I got one million questions to ask

But most importantly, I keep thinking about one thing (which is quite philosophical):

How people figure out what region in the DNA to study???

Say for example, I want to know whether a gene "A" has changes in histone modification in a certain type of cancer

And so I have some samples of liver cancer and normal tissue

I hypothesis that gene "A" is turned on in liver cancer

So I want to see if the histone near the promoter of gene "A" has more H3K4-tri-me and less H3K27-tri-me in cancer compared with normal tissues.

Now there's a problem....WHERE should I look???

For example, after I do CHiP using H3K4me3 and H3K27me3 Antibody to enrich the DNA, what should I do next if I don't know WHERE in the promoter (or even more upstream) to LOOK?

Design primers for PCR every 100bp? or do Southern??

Can anyone help me to understand more about the experimental flow???

Protocol for histone acid extraction for Western blot

Mon, 22 Aug 2005 01:54:39 -0700

hello!

desperately need some good protocol for histone acid extraction (for western blot)!

thanks in advance

Help with making positive control

Wed, 30 Jul 2008 22:14:56 -0700

I am preparing a postivie control for CpG methylated mouse genomic DNA.

I treated SssI with mouse genomic DNA purified manually.
Reaction condition like this:

gDNA 5ug
SssI methylase 10U (2.5uL)
SAM 320 uM
NEB buffer #2
water to total 50uL

4hrs at 37'C

I'd like to confirm if this DNA can be used as a globally CpG methylated positive control.
Can anybody help me how I could break through here?

Thank you.

nuclei isolation from Drosophila S2 cells

Thu, 07 Aug 2008 01:33:22 -0700

Hello,
I have just starting my PhD and I will start working with flies,which I have never done before.
I was looking for a protocol for nuclear run-off assay in S2 cells.
First trials I will try to extract nuclei.
I would be glad if someone has a working protocol for nuclei isolation in S2 cells.

Thanks.