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There have been 27 items by jamestoon1 (Search limited from 24-September 19)



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#146996 any journal recommendation?

Posted by jamestoon1 on 20 December 2012 - 12:41 AM in Dissertation and Paper Writing

any of the PLOS group? Don't know about publishing costs though.

Way too expensive...

  • PLOS Biology US$2900
  • PLOS Medicine US$2900
  • PLOS Computational Biology
    US$2250
  • PLOS Genetics US$2250
  • PLOS Pathogens US$2250
  • PLOS Neglected Tropical Diseases
    US$2250
  • PLOS ONE US$1350

Anyone has other recommendation? Posted Image



#146961 any journal recommendation?

Posted by jamestoon1 on 19 December 2012 - 10:31 AM in Dissertation and Paper Writing

Hi all.

I am looking for a journal
- with an ISI impact factor of around 2.0 - 2.5
- with low or no publication fee (my advisor is not willing to sponsor any amount greater than USD 200)
- in the area of medical science/cancer genetics/cancer molecular biology/colorectal cancer or other related areas

Any recommendation?



#146963 Question about RNA and expression

Posted by jamestoon1 on 19 December 2012 - 10:48 AM in -Molecular Biology-

We all know that RNA expression is tissue-specific.
So in many studies, researchers analyze the RNA expressions of certain genes in affected tissues and compare them to matched normal tissues.
For example, some breast cancer researchers would investigate the expression of BRCA1 in cancerous and noncancerous breast tissue, so that they can know whether deregulated BRCA1 expression occurs in breast cancer.
These are what I understand from my undergraduate course.

But, recently I came across quite a lot of studies, which use blood or salivary RNA to study gene expression of tissue-specific diseases such as breast cancer.
I fail to figure out the rationale of using blood or salivary RNA instead of RNA from the tissue to study gene expression.
Could someone please enlighten me on this?



#154214 Question about RNA and expression

Posted by jamestoon1 on 25 April 2013 - 05:18 AM in -Molecular Biology-

Cells from cancer tend to get into blood stream so you can get the specific genes that are normally expressed only in one type of tissue and overexpressed in cancerous tissue from blood. But you need normally expressed gene as well for reference.

Wow, thanks for the reply after so long.
Then how about RNA from saliva?



#146997 Question about RNA and expression

Posted by jamestoon1 on 20 December 2012 - 12:44 AM in -Molecular Biology-

Well, they are easiest to collect...

Also just found this via google http://iranpath.org/...71209000055.pdf , a review where they also discuss, starting from p.7, the similarities between breast tissue and the salivary glands and the respective fluids, although it seems to focus on proteins rather than RNA...


Oh, thanks for the reply.
Anyone else could tell me why RNA from blood/saliva is used instead of RNA from tissue?



#148548 Is my RNA degraded?

Posted by jamestoon1 on 22 January 2013 - 02:55 AM in Molecular Biology

In our lab, we have observed that with kits that use spin columns is normal to loose the smaller RNAs (they are not retained in the column), while those are still there with Trizol.

Back to your picture, to me it looks like you do have some degradation, also may have some DNA contamination (top of the gel, still in the well). It also looks like the gel is maybe overloaded and what I think is degradation (smeary bit between distinct bands) might just be due to too much RNA.

But I have read somewhere that if we isolate RNA with Trizol, the RNA integrity tends to be lower than isolating with RNeasy kit.
So if I need both small RNA and high integrity RNA for my work, based on your experience, would you suggest me to use Trizol or RNeasy?

I loaded 4ul RNA into the gel, and I only came to know later that for RNA, 2ul is sufficient, so most probably the smear is due to overloading of RNA.
But I'll run gel electrophoresis again after this.
DNA contamination is not unexpected because I did not use DNase this time (because this is only a trial experiment of mine).
I have failed several times in the past for my RNA isolation, and this is the first time I succeeded.
It turned out that my previous failed attempts was due to insufficient grinding of the tissues.



#148482 Is my RNA degraded?

Posted by jamestoon1 on 21 January 2013 - 06:03 AM in Molecular Biology

Thanks for the reply.



#148427 Is my RNA degraded?

Posted by jamestoon1 on 19 January 2013 - 10:11 PM in Molecular Biology

Degradation presents as a smear. Bands are other rRNA bands

Thanks for the reply.
But I have seen other people's RNA gel image, and most of them got something like this (only 2 clear bands without those smaller bands):
others.JPG
So why are those other rRNA bands present only in my samples?
Does the presence of those other rRNA bands indicate something good or something bad?



#148407 Is my RNA degraded?

Posted by jamestoon1 on 19 January 2013 - 09:40 AM in Molecular Biology

Hi.

I isolated RNA from human tissue samples using RNeasy.
I electrophoresed the RNA obtained and this is what I got:
is my rna degraded.JPG
P.S. 100bp DNA ladder was used instead of RNA ladder because we don't have RNA ladder in our lab.

From the gel image, two clear bands can be seen (supposed to be 18S and 28S rRNA).
But there are also some smaller bands down there.
Does this indicate RNA degradation or something else?

P.S. The tissue samples were preserved in RNAlater for 1 month before RNA isolation.

P.S. We don't have Bioanalyzer in the lab, so I cannot analyse it in the instrument.

P.S. I did not check the purity and concentration of the RNA because the only spectrophotometer in our lab has been send for repair.



#156396 How to calculate D' and R2 for linkage disequilibrium

Posted by jamestoon1 on 11 June 2013 - 10:26 AM in Bioinformatics and Biostatistics

Supposed i have 2 polymorphisms, SNPA and SNPB.
Individuals who carry the wildtype genotype for SNPA also carry the wildtype genotype for SNPB.
Individuals who carry the heterozygous genotype for SNPA also carry the heterozygous genotype for SNPB.

Individuals who carry the mutant genotype for SNPA also carry the mutant genotype for SNPB.

It is pretty obvious that the two polymorphisms are in complete linkage disequilibrium, right?
So the D' should equal to 1.

But, how do I calculate that?

I found a web-based software which seems to be easy to use, but I don't know what number should I key in onto each cell --> http://www.oege.org/software/cubex/

Now supposed I have 1000 wildtype, 800 heterozygous and 600 mutants for both polymorphisms, what number shall I put in the cells?

I tried the following (based on the frequencies of my genotypes):
2000---1800---1600
1800---1600---1400
1600---1400---1200

But I got a D' value of -0.011 instead of 1.

Can anyone help?

You may recommend other softwares or simple methods for calculating the D' and r2.

Thanks in advance.



#156446 How to calculate D' and R2 for linkage disequilibrium

Posted by jamestoon1 on 12 June 2013 - 09:33 AM in Bioinformatics and Biostatistics

Nobody answered, but never mind, I already know the answer.
Supposed to fill as follows:

1000---0---0
0---800---0
0---0---600



#145742 What's wrong with my PCR

Posted by jamestoon1 on 23 November 2012 - 10:31 AM in PCR, RT-PCR and Real-Time PCR

I tried to amplify a region, but here's what I got: one.JPG

The band size of interest is 200bp and I got a lot of smear (should i call it smear because it has specific discrete pattern).


Here the details of the PCR:

2X PCR master mix ---> 10 ul
Forward primer (10 pmol) ---> 0.5 ul
Reverse primer (10 pmol) ---> 0.5 ul
Template DNA ---> 0.5 ul

I have tried diluting the template DNA, but I still get all those smears (i.e. when I diluted the template, both the band of interest and the smear become faint)

I don't think the problem lies on the primers, because
(1) there has been many previous publications utilizing this primer pair
(2) i thought probably my primers degraded or something like that, so I tried to order new sets of primer (same sequence, but newly synthesized one) from different companies

-----------------------------------------------------------------------

Initial denaturation (95°C, 2 minutes)

35 cycles:
Denaturation (94°C, 30 seconds)
Primer annealing (60°C for 45 seconds)
Elongation (72°C, 1 minute)

Final extension (72°C, 5 minutes)

-----------------------------------------------------------------------

Can anyone help? Thanks in advance~



#145768 What's wrong with my PCR

Posted by jamestoon1 on 23 November 2012 - 08:45 PM in PCR, RT-PCR and Real-Time PCR

Hi. Thanks for the reply. It was 100 bp ladder.
The band of interest was there, but the primer dimers are soooooo much more pronounced than the band of interest.
I have also tried (1) touchdown pcr, (2) increasing the annealing temp, (3) adjusting (increasing/decreasing) the duration of denaturation, annealing, extension, but all gave me similar results.
Can anyone advise? Posted Image



#145786 What's wrong with my PCR

Posted by jamestoon1 on 24 November 2012 - 10:16 AM in PCR, RT-PCR and Real-Time PCR

Hi James,

Did you run a No template control along with this PCR? I am just guessing if these smears are directly from your template DNA. What is the source of DNA?


To check the primers you can reduce the primer concentration too e.g. down to 0.1 uM (you have now a concentration of 0.25 uM if you have 20 ul total volume per tube). If the smear becomes less it's perhaps a primer-dimer problem (they can also be amplified, if they overlap in a way that the polymerase can attach; this can happen with badly designed primers).
And try out AmeyaP's idea of course.


Thanks for the suggestions. Will try them and let u know the result. Posted Image



#148062 isolating DNA from cells in suspension

Posted by jamestoon1 on 14 January 2013 - 09:41 PM in Molecular Biology

Thanks for the feedback!



#147943 isolating DNA from cells in suspension

Posted by jamestoon1 on 13 January 2013 - 06:31 AM in Molecular Biology

It would be so that you can concentrate your cells into a much smaller volume to facilitate the DNA extraction.

Oh..so if the protocol recommends us to resuspend the cell pellet into 200ul of buffer, and I have an initial cell suspension volume of 200ul, does it mean that I don't have to perform the centrifugation and resuspension?



#147936 isolating DNA from cells in suspension

Posted by jamestoon1 on 13 January 2013 - 02:52 AM in Molecular Biology

When isolating DNA from cells in suspension, most if not all the protocols I have come across recommend centrifugating the suspension at the very beginning to obtain a cell pellet, followed by resuspension of the pellet in either water or PBS or other buffers.

May I know what is the purpose of obtaining this cell pellet?

Can we just vortex the suspension and use it directly without pelleting the cells?



#136724 smearing below band of interest

Posted by jamestoon1 on 30 June 2012 - 09:59 PM in PCR, RT-PCR and Real-Time PCR

i am doing touchdown pcr with the following condition:

98'C 30 sec initial denaturation

10 cycles:
98'C 5 sec denaturation
60'C 5 sec annealing
72'C 5 sec extension

25 cycles:
98'C 5 sec denaturation
59'C 5 sec annealing
72'C 5 sec extension

72'C 2 min final extension

my band of interest is 200bp
i got the band
but there are smearing below the band:
Attached File  untitled.bmp   468.18KB   1402 downloads

what has gone wrong and how should i rectify the problem?

FYI, my pcr master mix contain 18.5ul master mix, 0.5 ul forward primer, 0.5 ul reverse primer, 0.5 ul DNA.



#136760 smearing below band of interest

Posted by jamestoon1 on 02 July 2012 - 03:10 AM in PCR, RT-PCR and Real-Time PCR

Given the 98 denature, I assume you are using Phusion. Phusion master mixes are usually 2x master mixes. You should double check to make sure you are not missing the added water.


Yes, it's Phusion. It was a typographical error. What I used was 10ul master mix plus 8.5ul water.



#136761 smearing below band of interest

Posted by jamestoon1 on 02 July 2012 - 03:14 AM in PCR, RT-PCR and Real-Time PCR

How much of your PCR volume did you load on the gel? It looks very overloaded to me. The smearing below your product could be primer dimer.

Just out of interest, can I ask what the rationale for using those cycle conditions is? It seems odd to me to have two annealing temperatures that are SO close.

I loaded 3ul.
60'C is the highest achievable temp. I mean, after 60'C, i can no longer get any pcr product. so i chose to use tat one as the starting temperature.
and i chose 59'C because from my optimization experiments, 59'C can result in multiple bands, so i would not go below that temp.
there should be no problem with primer design. i have BLASTed and have checked with a few softwares. Moreover this is the primer pair obtained from literature. A lot of people have used this primer pair.



#136228 problem of restriction enzyme digestion

Posted by jamestoon1 on 21 June 2012 - 09:23 AM in Molecular Cloning

can anyone help me??



#136188 problem of restriction enzyme digestion

Posted by jamestoon1 on 20 June 2012 - 07:09 PM in Molecular Cloning

hi all,

i am doing SNP genotyping.
my pcr product is 280 bp
if the subject is homozygous wildtype, the RE will not cut, so i will get a single 280 bp band

if the subject is homozygous variant, the RE will cut, and i will get a 240 bp band and a 40 bp band
if the subject is heterozygote, i will get all 280 bp, 240 bp and 40 bp bands

now i have one problem
i cannot be sure whether the RE cuts or not
for some samples, the 240 bp band seems to be present but the band is very very faint (indicated by the red arrows in the following gel)
sssa.JPG

so, my question is, how should i interpret these? are they heterozygote or homozygous wildtype?
what can i do to improve RE cutting?
the protocol mention use 1 unit of enzyme and incubate for 1 hour. i have already tried increase the enzyme to 10 units and incubation for up to 6 hours, but the appearance of the bands are still unclear.
i have several thousands of samples to run, so it would be infeasible to send all for sequencing.



#136253 problem of restriction enzyme digestion

Posted by jamestoon1 on 21 June 2012 - 07:18 PM in Molecular Cloning

bump



#155958 accidentally used denatured ethanol for DNA and RNA - what to do?

Posted by jamestoon1 on 02 June 2013 - 08:36 PM in Molecular Biology

Thanks for all the replies.
Due to shortage of time (I need to do some trial tests today), I proceeded with DNA and RNA isolation with the reagents added with denatured ethanol.
The spectrophotometric readings were good - the yield and purity were comparable to DNA/RNA isolated with the reagents with non-denatured ethanol.
Let's hope any impurities that may be present would not interfere with downstream applications.



#155913 accidentally used denatured ethanol for DNA and RNA - what to do?

Posted by jamestoon1 on 01 June 2013 - 12:55 PM in Molecular Biology

My DNA and RNA isolation kits require me to dilute some of the buffers with absolute ethanol.
I added denatured ethanol to all the necessary buffers, then only I came to realize that we should use non-denatured ethanol for molecular biology applications because denatured ethanol contains impurities.
What should I do now?
Can I proceed with DNA and RNA isolation using the reagents?
If not, my supervisor is going to kill me OMG!!!




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