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Forum: Epigenetics 10-30-2013, 01:50 AM
Replies: 17
Views: 7,481
Posted By C.R.
Yes it is from NEB. For the gel I used 3g DNA,...

Yes it is from NEB. For the gel I used 3g DNA, 5l buffer 4, 2l MspI in a total volume of 50l. 1/3 of the digest was directly used for the gel without phenol-extraction. The gel is 0.7% Agarose in...
Forum: Epigenetics 10-29-2013, 09:08 AM
Replies: 17
Views: 7,481
Posted By C.R.
I am not too sure about your image, because the...

I am not too sure about your image, because the undigested control is missing. In fact I think, that you can see a smear of fragments on your gel picture.

Please have a look at the attached...
Forum: Epigenetics 10-28-2013, 10:29 AM
Replies: 17
Views: 7,481
Posted By C.R.
Yes, I was also very confused when I looked at my...

Yes, I was also very confused when I looked at my first digest on a 2 percent gel, since I thought that it did not work. Basically what I saw was an accumulation of long fragments which made me think...
Forum: Epigenetics 10-28-2013, 10:13 AM
Replies: 17
Views: 7,481
Posted By C.R.
Hey, the digest just works so good, that I...

Hey, the digest just works so good, that I already omit to check it on a gel to safe DNA for some samples. I also used NEB and Fermentas and prepared Libaries with both. If you really want to see the...
Forum: General 10-15-2013, 01:48 AM
Replies: 4
Views: 2,319
Posted By C.R.
I would say your are correct. If you focus on...

I would say your are correct. If you focus on CpGs then you should stay with the CG which is the same in forward and reverse orientation.
Forum: Epigenetics 09-24-2013, 05:55 AM
Replies: 17
Views: 7,481
Posted By C.R.
here is an example:...

here is an example: http://www.zymoresearch.com/epigenetics/dna-methylation/methylated-dna-standards/human-methylated-non-methylated-dna-set

You probably used a 2% agarose gel to get a nice...
Forum: Bioinformatics 06-04-2013, 01:11 AM
Replies: 1
Views: 613
Posted By C.R.
In my understanding it is like this: the sequence...

In my understanding it is like this: the sequence you provide contains one CpG site which can be sequence from forward and reverse strand. However, if you want to calculate the methylated cytosine...
Forum: Illumina/Solexa 04-09-2013, 01:48 AM
Replies: 5
Views: 2,187
Posted By C.R.
I did not test the gel free protocol yet. I...

I did not test the gel free protocol yet. I always had adapter dimers in the final library when the gel was running too fast and I never had this problem with the old PE adapters
Forum: Illumina/Solexa 04-05-2013, 06:41 AM
Replies: 5
Views: 2,187
Posted By C.R.
I never used 5ng DNA but TrueSeq Adapters in...

I never used 5ng DNA but TrueSeq Adapters in generall are not a problem. OK, still we get more Adapter dimer. Next time I will use less Adapter. This is even more important when you use only 5ng. But...
Forum: Illumina/Solexa 04-05-2013, 06:17 AM
Replies: 3
Views: 1,662
Posted By C.R.
We bought the PhiX from Illumina. I am not sure,...

We bought the PhiX from Illumina. I am not sure, if there is an indexed one available. At least in the past this was not the case. This means that in fact we cannot exclude that some reads may come...
Forum: Bioinformatics 02-28-2013, 02:38 AM
Replies: 7
Views: 2,639
Posted By C.R.
You may try...

You may try http://bisearch.enzim.hu/?m=genompsearch
Forum: Bioinformatics 11-09-2012, 02:47 AM
Replies: 15
Views: 2,068
Posted By C.R.
I would check the overall performance of this...

I would check the overall performance of this lane in the Illumina Sequencing Analysis Viewer. I guess that the barcode read will have some errors which results in the failure of the demultiplex. You...
Forum: Illumina/Solexa 09-18-2012, 06:09 AM
Replies: 6
Views: 2,705
Posted By C.R.
well, I did some RRBS sample prep. using the...

well, I did some RRBS sample prep. using the TruSeq Adapter. These libraries have been sequenced and it looks OK. We needed to use custom made primers very soon, since these were limited in the...
Forum: Illumina/Solexa 09-18-2012, 02:27 AM
Replies: 3
Views: 1,662
Posted By C.R.
we do not spike our ChIP-Seq Libraries, but some...

we do not spike our ChIP-Seq Libraries, but some transcription factor ChIPs may benefit from this. However, we are using the 40-50% spike-in for RRBS libraries which are really challenging when...
Forum: Illumina/Solexa 09-18-2012, 01:20 AM
Replies: 6
Views: 2,705
Posted By C.R.
I am not sure if you still can buy the methylated...

I am not sure if you still can buy the methylated PE Adapters from Illumina but they worked good in our hands. We successfully used the TruSeq Adapters for RRBS, but you have to adjust the Gel...
Forum: Bioinformatics 08-30-2012, 02:39 AM
Replies: 2
Views: 1,961
Posted By C.R.
Fastq export and demultiplexing is usually...

Fastq export and demultiplexing is usually performed with the Illumina CASAVA software: http://support.illumina.com/sequencing/sequencing_software/casava.ilmn.
Forum: Bioinformatics 08-07-2012, 05:41 AM
Replies: 2
Views: 3,400
Posted By C.R.
Yes, this is the correct way. If you store the...

Yes, this is the correct way.
If you store the executable at a different place make sure that you provide the full path to it:

/pathToLiftover/liftOver oldFile /pathToLiftover/map.chain newFile...
Forum: Illumina/Solexa 05-22-2012, 02:08 AM
Replies: 3
Views: 1,746
Posted By C.R.
Hi, thats an interesting enzyme. It looks like...

Hi, thats an interesting enzyme. It looks like that the 'improved representation of AT-rich sequences' introduces another bias. Maybe you should compare your data with an Agilent Pfu Turbo Cx derived...
Forum: Sample Prep / Library Generation 02-02-2012, 04:36 AM
Replies: 3
Views: 3,081
Posted By C.R.
I do not think that glycogen is the major...

I do not think that glycogen is the major problem, since I use it as well during ethanol precipitation after phenol extraction in an illumina library prep. protocol. But I cannot exclude that there...
Forum: Bioinformatics 08-03-2011, 10:26 AM
Replies: 9
Views: 15,431
Posted By C.R.
Hi, I usually increase the RAM used, change the...

Hi,
I usually increase the RAM used, change the validation stringency and define a tmp dir.

My example will look like this:
java -Xmx4g -jar ~/bin/picard/MarkDuplicates.jar...
Forum: Bioinformatics 05-18-2011, 08:48 AM
Replies: 4
Views: 2,539
Posted By C.R.
Well there is something which is pretty much what...

Well there is something which is pretty much what you asked for: http://medgen.ugent.be/methBLAST/
I never used it myself, but maybe it is useful for you. But if I were you I would try to get it...
Forum: Bioinformatics 04-05-2011, 04:53 AM
Replies: 17
Views: 9,279
Posted By C.R.
I strongly agree. This is a big problem and...

I strongly agree. This is a big problem and Illumina does not pay attention to it. In general my libraries are OK, since it worked for one test run on a Genome Analyzer. Now I got 5 RRBS samples...
Forum: Epigenetics 04-01-2011, 01:32 AM
Replies: 5
Views: 3,114
Posted By C.R.
I digest 1g DNA and use a standard 0.7% agarose...

I digest 1g DNA and use a standard 0.7% agarose gel. I see a smear starting from uncut = approx. 10,000 to 100-200. The intensity below 200 bps gets lower. With 1% it should already look much better...
Forum: Epigenetics 03-28-2011, 08:43 AM
Replies: 5
Views: 3,114
Posted By C.R.
I think the trick is to use an appropriate gel...

I think the trick is to use an appropriate gel for checking. On a high percentage gel the DNA looks uncut, but on lower percentage gel my samples look similar to the the example from the Zymo web...
Forum: Illumina/Solexa 03-23-2011, 12:45 PM
Replies: 6
Views: 3,276
Posted By C.R.
Hi, which protocol do you actually you refer to,...

Hi, which protocol do you actually you refer to, since there are several publications out for shotgun bisulfite sequencing. One example: http://www.ncbi.nlm.nih.gov/pubmed/21289626
There the...
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