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pcrman's Content

There have been 95 items by pcrman (Search limited from 21-July 18)



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#169525 Finding upstream sequence and e-PCR

Posted by pcrman on 20 July 2014 - 11:18 PM in -Molecular Biology-

In this post, I provided steps on how to find upstream (promoter) sequences. The instructions apply to worm upstream sequences too.

 

Hope that helps.




#169522 prediction of microRNA targets using the sequence

Posted by pcrman on 20 July 2014 - 10:55 PM in siRNA, microRNA and RNAi

You can use command line miRanda program to run target prediction.

 

1. download the program from this page http://www.microrna....getDownloads.do (at the bottom)

2. format your miRNA or small RNA sequences in FASTA format (you can give any unique ID to each sequence. The format will look like this:

>seq1

ccaaauugaaguucau

>seq2

ccagagauucaguagau

 

3. download sequences again which targets are to be predicted as fasta file. For example you can download all 3UTR sequences of an organism using BioMart tool

4. run the program in a linux environment, or a virtual linux environment in windows or mac.




#169258 DNA yields after Bisulfite Treatment

Posted by pcrman on 08 July 2014 - 09:15 AM in DNA Methylation and Epigenetics

Hi, I am new to this forum and doing BS conversion with Epitech Bisulfite conversion kit. I am using 10ug DNA but i am getting only 50ng DNA after BS conversion. I am doing the quantification with Qubit  from Invitrogen.  Please suggest me to get high amount of DNA after conversion.

 

Thanx

Hi Rama,

 

I think you cannot use too much starting DNA in order to get high DNA yield. What amount of DNA does the kit suggests you to use? Using too much starting DNA can lead to incomplete conversion of unmethylated cytosines to uracils.   We don't usually measure DNA concentration after conversion, and to see amplification, we do two rounds of PCRs.




#167106 CRISPR vitro system

Posted by pcrman on 17 April 2014 - 10:33 AM in Molecular Biology

Hey all,

 

Does any know if there is a company or a group that is selling or able to provide the CRISPR-CAS9 protein?

I would like to use the CRISPR system invitro to cut DNA. I can't use restriction enzymes.

 

Thanks

That is a very interesting idea. I am not sure whether earlier papers studying CRISPR have tried this like RNAi people have for in vitro RNAi. In this in vitro system, you need to add guide RNA which could be chemically synthesized.

 

Edit: Just found a paper: In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex.  




#167105 Nested BSP Primer Design

Posted by pcrman on 17 April 2014 - 10:28 AM in DNA Methylation and Epigenetics

In general, BSP primers should be longer than those for regular PCR, definitely not shorter.  You may not need nested pairs, just re-amplify using the same pair. 




#167104 finding a reference for a formula

Posted by pcrman on 17 April 2014 - 10:21 AM in Molecular Biology

The calculation has to be based on the sequence. There is an online tool allowing you to do that http://endmemo.com/bio/dnacopynum.php. maybe you can cite the website.

 

This paper may also help:

Determination of the exact copy numbers of particular mRNAs in a single cell by quantitative real-time RT-PCR




#167066 Setting up bisulfite sequencing in lab

Posted by pcrman on 15 April 2014 - 02:02 PM in DNA Methylation and Epigenetics

For each sample, how many sequence regions are you going to examine?  

 

You don't need to sequence unmodified version of every sample. the purpose of sequencing a few of such sample is to let you know whether your modification procedure can successfully convert unmethylated cytosines. Comparing sequencing results from bisulfite modified DNA to reference sequences (from genome database) will tell you where cpgs are.

 

I have not used any analyzing program. I just use simple sequence alignment program to align the reads.  




#167065 Subsequent DNA and siRNA transfection

Posted by pcrman on 15 April 2014 - 01:48 PM in siRNA, microRNA and RNAi

We mostly use invitrogen's RNAiMax which has low toxicity.




#166958 Setting up bisulfite sequencing in lab

Posted by pcrman on 11 April 2014 - 09:25 AM in DNA Methylation and Epigenetics

How many samples are you going to analyze? My point is that if you don't have a lot of samples and many years of project to run, it is not worth setting up your own sequencing center since nowadays sequencing have become very cheap (~$5/sample)

 

As Phage has told, you will need a primer pair that only amplify bisulfite modified DNA to discriminate against unmodified DNA (for most conversion, there is likelihood of incomplete conversion). To do so, the primers need to be designed in regions which must contain a certain number of non-cpg Cs (e.g., at least 5), but do not contain any CpG Cs. If CpG Cs are unavoidable, you can use degenerate primers to cover both possibilities for the C, that is methylated (use a C) or unmethylated (use a T) C.

 

For bisulfite sequencing, the most difficult part is not at sequencing, but at PCR reaction! After PCR, there are two strategies for sequencing: direct sequencing and sequencing after cloning.  the former, although quick, cannot usually give very clean data (you may get high background/noise if PCR products are not clean enough); the latter gives your cleaner data, but labor intensive (you need to sequence at least 10 colonies).

 

If you have further questions, please let us know. 




#166884 shRNA problem - Higher expression at the mRNA level !

Posted by pcrman on 09 April 2014 - 03:26 PM in siRNA, microRNA and RNAi

This phenomenon can now probably be explained by the RNAa mechanism. In C. elegans, 22G-RNAs (which are secondary RNAs derived from piRNAs) antisense to endogenous mRNA are loaded by the CSR-1 Argonute protein to form a complex which then enters the nucleus. the 22G-RNA/CSR-1 complex binds to nascent mRNA to promote the transcription of mRNA epigenetically. You can read the following papers:

 

The C. elegans CSR-1 argonaute pathway counteracts epigenetic silencing to promote germline gene expression.

Seth M, Shirayama M, Gu W, Ishidate T, Conte D Jr, Mello CC.

 

Global effects of the CSR-1 RNA interference pathway on the transcriptional landscape.

Cecere G, Hoersch S, O'Keeffe S, Sachidanandam R, Grishok A. 




#166883 Reference genes in miRNAs

Posted by pcrman on 09 April 2014 - 03:16 PM in siRNA, microRNA and RNAi

No, what you described is not correct. You need to have at least one internal control such as sn/snoRNAs. Please read this.




#166882 Lentivirus Packaging

Posted by pcrman on 09 April 2014 - 03:10 PM in General Lab Techniques

Do you mean GFP is low after you infect cells with your viral particles? If yes, you can improve this by using higher concentration of virus and fresh ones. Do you see good GFP during packaging?




#166881 EMSA Shifted band in Free probe only lane

Posted by pcrman on 09 April 2014 - 03:05 PM in General Lab Techniques

Sorry for the late reply. Hope you have already solved the problem. 

 

The gel patterns look very weird to me. since you also got the nonspecific shift (band) in your free probe lane, I doubt it is a shifted band. It appear to be a higher sized DNA band. Is It possible when you do annealing, the oligos not only formed duplex DNA, but also concatenated to give a longer dsDNA.  




#166880 Ubiquitin promoter cloning

Posted by pcrman on 09 April 2014 - 02:55 PM in Molecular Cloning

I think your question is too specific to be answered. What is FUW plasmid, what ubiquitin promoter sequence is already in it? 




#166879 Subsequent DNA and siRNA transfection

Posted by pcrman on 09 April 2014 - 02:53 PM in siRNA, microRNA and RNAi

We have done transfection with plasmid and siRNA. We transfect them either sequentially (first plasmid and then siRNA as you planned) or in combination. Transfection efficiency is not a big issue.  




#166806 Methylation status analysis of non CpG sites.

Posted by pcrman on 08 April 2014 - 09:32 AM in DNA Methylation and Epigenetics

it is tricky to use MSP to check non-cpg C methylation since there is not many place you can put your primers in without including a non-cpg C. 




#166804 Preparing overlapping bisufite sequences for methylation analysis

Posted by pcrman on 08 April 2014 - 09:30 AM in DNA Methylation and Epigenetics

 

 

my question is do I cut out the sequences so that there are no overlapping regions before analysis?

I understand that you meant the analysis phase, right? You don't need to cut out the overlapping regions. actually by overlapping regions, you can obtain more representative data, i.e., an overlapping region is represented by 20 clones instead of just 10. So I think the data should be included and presented. 




#166802 purpose of 65 degrees incubation overnight after beads elution for CHIP

Posted by pcrman on 08 April 2014 - 09:26 AM in DNA Methylation and Epigenetics

that is to reverse the crosslink done by formaldehyde at the beginning. 




#166801 Can ChIP sequencing be used to make exploratory findings about histone methylati

Posted by pcrman on 08 April 2014 - 09:24 AM in DNA Methylation and Epigenetics

So your question is to find genes differential expressed in cancer via epigenetic mechanisms and you want to approach this question by ChIP-seq. You don't need to go after all possible histone modifications, and can focus on a few such as H3K4m3 (active promoter), H3K36me3 (active transcription in gene body), H3k9me3 and H3K27me3 (repressive marks), To  get a sense of gene expression/transcription levels, you can also include RNAP II in ChIP-seq, alternative, you have to do a cDNA microarray or RNA-seq.

 

but all these ChIP-seq results just give a correlation between gene expression and epigenetic status, cannot really answer your question why some genes are deferentially expressed. Histone modification changes may just reflect the status of gene expression. If you really want to dive deep into this kind of questions, you have to take genetic factors, transcription factor binding and DNA methylation into consideration.    




#166799 fixation and methylation

Posted by pcrman on 08 April 2014 - 09:03 AM in DNA Methylation and Epigenetics

Nope. But keep in mind, since DNA from paraffin blocks is already degraded, and will be further fragmented during bisulfite conversion, amplicons size should be kept to a minimum when designing primers. 




#166798 Bisulfite/sequencing-based Methylation Analysi

Posted by pcrman on 08 April 2014 - 08:58 AM in DNA Methylation and Epigenetics

In most cases, 35 cycles are not enough. Are you doing MSP or BSP? If it is for BSP, you can reamplify your first round products.  




#164825 How to view the encapsulated cells under microscope?

Posted by pcrman on 05 February 2014 - 01:19 AM in Microbiology

What are encapsulated cells? 




#164823 A help for siRNA experiment

Posted by pcrman on 05 February 2014 - 01:15 AM in siRNA, microRNA and RNAi

Hi madelingirly,

 

here is a list of things you need to think about when trouble shooting:

1) Transfection efficiency: how well have the siRNAs been transfected into your cells? In general, siRNA transfection efficiency is not a big concern, but may vary in different cells. How about you purchase a fluorescence labeled siRNA as a control for transfection efficiency?

2) I don't think IF is a good way of accessing gene knockdown. IF itself can give you false positive signal. Try RT-PCR and western.

3) Do you know the basal expression of the target genes in your cells? Are they already expressed low?




#164417 Suggestions on software used to open large text files

Posted by pcrman on 22 January 2014 - 11:22 AM in Bioinformatics and Biostatistics

I don't know any mac program, but for PC, textpad can open large text files. Alternatively, you can install virtual Linux OS in Mac OS. 

 



#163300 Determining CpG methylation and effect on gene expression

Posted by pcrman on 10 December 2013 - 10:22 PM in Bioinformatics and Biostatistics

Have you read a recent paper on this topic? Please see my post in this thread: http://www.protocol-...hylation-study/





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