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vetticus3's Content

There have been 143 items by vetticus3 (Search limited from 22-February 19)



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#165756 Please please help me with my Phusion PCR.

Posted by vetticus3 on 05 March 2014 - 07:35 AM in PCR, RT-PCR and Real-Time PCR

a normal phusion pcr reaction is this:

 

 

Initial Denaturation 98°C 30 seconds

 

25-35 Cycles

98°C - 5-10 seconds

45-72°C - 10-30 seconds

72°C - 15-30 seconds per kb

 

Final Extension 72°C 5-10 minutes

 

Hold 4-10°C  

 

generally, the 45-72°C step you'd begin with 55°C

 

what program did you use to design the primers?  something went very wrong somewhere.




#165755 how to hasten real-time PCR amplifications

Posted by vetticus3 on 05 March 2014 - 07:31 AM in PCR, RT-PCR and Real-Time PCR

seconding the more cDNA.




#165754 transcription factor binding site prediction, identification

Posted by vetticus3 on 05 March 2014 - 07:24 AM in Bioinformatics and Biostatistics

Ok, i'll have a go at answering what i can...

 

so, you need the sequence of the promoter of runx3.  to search for the promoter region, you need to go to NCBI and search the nucleotide database using the goi name, promoter, or 5 flanking. this will give you a lot of sequences, and you need to go through them to see which one is appropriate for you.  the promoter sequences in GenBank are submitted by researchers and are usually characterized.

or, you could go to ensembl genome browser... for runx3:  http://www.ensembl.o...226002-25291612

here you should be able to see the different introns and exons.

Or my favourite, gene cards, and look up the promoter sequence from there.  http://www.genecards...p.pl?gene=RUNX3

 

it's possible for genes to have multiple promoter sites... especially if they are alternatively spliced. i always thought runx3 had two promoters, see the Groner Lab for info on this.  if you have identified another one, you should discuss it with your supervisor. 

 

once you have the sequence, you need to have a general idea of the transcription factor binding sites.  this means a literature search... and for pu.1 the putative binding site is GAGGAA.  klf4 is RCRCCYY or CACC.

 

i then used to manually search for the binding site sequence in my gene of interst.  so, I have no idea about which software to use.  manually= with your eyes. (for a couple of genes, this is ok.... for all of them, it's a problem...but you're only looking at 1).

also keep an eye open for partial binding sites etc, as nothing is set in stone.

 

to validate... do a ChIP (wet lab).  nothing you do in silico will validate actual binding.  until you can show that the transcription factors are binding to the chromatin (via ChIP), all you have is a "potential" binding site.

 

the ChIP-seq data you have should be global - it should contain all the places where the transcription factor binds.  from that, you should be able to see a pattern.  this will include other binding site sequences, of preferential binding areas.




#165748 grant - why should we give you this money? expain in 1000 words

Posted by vetticus3 on 05 March 2014 - 05:09 AM in Grantsmanship

The exact question is:

Upload a statement (1000 words max) explaining precisely how the Fellowship will enable you to undertake your research and contribute to your career and why the Fellowship is important to the funding of your research.

 

it's money from l'oreal, so i could say: because I'm worth it  cool.png

it's not enough cash for a salary... it's enought for a dozen antibodies.  alright, so i have to go on a bit of a spiel about women in science etc.

thanks people smile.png




#165745 fundraising - anyone wanna give some?

Posted by vetticus3 on 05 March 2014 - 01:40 AM in Chit Chat

wow... i didn't realise there wasn't a paypal option.  you could always send me the money in a envelope ph34r.png




#165715 grant - why should we give you this money? expain in 1000 words

Posted by vetticus3 on 04 March 2014 - 07:24 AM in Grantsmanship

Hi,

Help me please!!

I'm applying for a little grant, and they ask the usual questions (explain in 200 words to a layman your project, expain in 1000 words your project etc)... but they also ask for 1000 words why their grant is going to help me.

 

Because money is nice, and i can buy things with it.  989 words to go.

 

In all honesty, 1000 words seems a bit much to kiss up to them for a few thousand pounds.

What do they want to hear?

They have another section where I have to break down the costs of the things I'm going to buy... do i just reitterate that the money is going to help support my essential equipment and servies, and possibly be used in a conference... and that they are just so wonderful and lovely etc.

 

help?

 

V




#165714 fundraising - anyone wanna give some?

Posted by vetticus3 on 04 March 2014 - 06:01 AM in Chit Chat

HA... i think it's the UK.

I've been doing fun runs for the past 10 years, and this is the first time that I had to also provide extra fundraising.  You would think that the price of actually entering would be enough... nope.  Not enough.

 

So... gonna give me a couple of bucks?




#165680 fundraising - anyone wanna give some?

Posted by vetticus3 on 03 March 2014 - 06:23 AM in Chit Chat

Hi all,

 

I've been talked into doing a fun walk with my mum to help raise $$$ for breast cancer research.

If anyone has a spare $, and really want to give it to a breast cancer charity... of even if you have a spare $ and don't really mind giving it to a breast cancer charity... you can go here:  http://wtwalk.org/mo...nd2014/yvette-2

 

We're walking because, shhh, mum turned 77 this year, and well, there was no running option.

 

Please, if you've got a minute, we'd really appreciate it.

 

Cheers,

V

 

PS, yes, we're dressing up a super heroes.




#163393 Words, Words, Words

Posted by vetticus3 on 13 December 2013 - 02:15 AM in Chit Chat

awesome foursome

 

morning peeps :)




#163020 strange raw data -

Posted by vetticus3 on 02 December 2013 - 07:17 AM in PCR, RT-PCR and Real-Time PCR

in case anyone has this issue... it's the gain function on the program.

check with the user manual on how to adjust this.




#163012 venting - oh ffs... looking for work.

Posted by vetticus3 on 02 December 2013 - 03:03 AM in Venting and Counseling

No, I decided a while back, and I'm currently working at new job... in the UK.




#162961 venting - oh ffs... looking for work.

Posted by vetticus3 on 29 November 2013 - 02:34 AM in Venting and Counseling

I took job A. 

Dance of joy.... :D

 

Job B's PI tried to make me feel guilty about it... and I nearly caved, but, I'm better in the long run.




#162958 immunofluorescence - antibody choice for double staining

Posted by vetticus3 on 29 November 2013 - 02:01 AM in General Lab Techniques

That was my initial line of thinking...  thanks for the advice.

Best,

V




#162957 strange raw data -

Posted by vetticus3 on 29 November 2013 - 01:58 AM in PCR, RT-PCR and Real-Time PCR

Ah, but that the rub.  I've used these sample before, and had no problem with it.  Also, I found out last night, that if I use the same standard curve (which produces that flatline) on a different position, it turns to rubbish - way too much variability between samples.

Still, I've got a shift of 10'C, a melt curve that looks like bad modern art, and previously verified samples turning bad.

 

Has anyone had problems with software corruption?  or filters getting dirty?  Either it's me (not the samples... but actually me doing bad voodoo on the experiment) or it's the machine/software.




#162935 strange raw data -

Posted by vetticus3 on 28 November 2013 - 09:09 AM in PCR, RT-PCR and Real-Time PCR

Hi,

has any one had a problem where there are certain samples that reach the maximum threshold at the first cycle (ie, there is no amplification curve just a flatline at max levels).... but there is a product that is melting at the correct temperature?

To make matters a bit stranger... the samples which are looking "correct" have a melting curve that is about 10'C coller than normal... and are have a much later than normal ct?

 

This is a machine or software problem?

I have changed

primers

water

samples

re run samples that have previously worked

 

it is the same position for the max amplifications.... but still having everything else melting at a cooler temp... i have never had this happened before and it's driving me mad.

 

thanks,

v




#162926 immunofluorescence - antibody choice for double staining

Posted by vetticus3 on 28 November 2013 - 04:57 AM in General Lab Techniques

Hi,

 

I need some advice - I'm going to be doing a double IF staining on PFA fixed cells.  One antibody I have - anti rabbit... the other antibody I'm going to buy.

The problem is the best antibody is also anti rabbit. 

There is an alternative that is anti mouse, but it has only been tested (and guaranteed) in IHC.

So... should I buy the anti mouse that may or may not give a result?

or

should i buy the anti rabbit and do a full stain with 1 ab, and then repeat with the second (which i haven't done and i'm not confident that it would work)?

 

Opinions?

 

V




#162275 Literature quote guessing game

Posted by vetticus3 on 11 November 2013 - 03:47 AM in Chit Chat

honestly, i've just started a new job which doesn't give me much down time...

how about an occassional quote




#162172 Literature quote guessing game

Posted by vetticus3 on 07 November 2013 - 02:17 AM in Chit Chat

salman rushdie?  it sounds like something from satanic verses.  he's describing gabriel (or however the name is spelt in that book).




#160958 Literature quote guessing game

Posted by vetticus3 on 07 October 2013 - 12:26 AM in Chit Chat

New quote Tabs?




#160809 Literature quote guessing game

Posted by vetticus3 on 01 October 2013 - 02:27 AM in Chit Chat

Not quite... this book can make you feel a bit sick in the stomach.




#160808 Happy B-day V3 (Bioforum's most amazing nonagenarian)

Posted by vetticus3 on 01 October 2013 - 02:22 AM in Chit Chat

I did nothing exiting on the day: watched the final Breaking Bad, internet apartment shopping, and had a scary mole removed.

Though I did have a birthday month - went to Bruges, Hannover and Scotland... and i'm going back to Scotland tonight for a few days.  Haggis, yummm.




#160786 Happy B-day V3 (Bioforum's most amazing nonagenarian)

Posted by vetticus3 on 30 September 2013 - 08:58 AM in Chit Chat

Thanks peeps.

93 today, and I don't feel a day over 80 biggrin.png

 

That mouse is hypnotizing.blink.png




#160784 Literature quote guessing game

Posted by vetticus3 on 30 September 2013 - 08:56 AM in Chit Chat

Yes... Sartre.  Wanna guess the title?




#160763 Literature quote guessing game

Posted by vetticus3 on 30 September 2013 - 01:03 AM in Chit Chat

No, but the author is French.




#160747 Literature quote guessing game

Posted by vetticus3 on 29 September 2013 - 03:57 AM in Chit Chat

Thanks smile.png

 

Ok, so a nice easy quote.  No clues yet, because it is so easy...

 

I suppose it is out of laziness that the world is the same day after day.





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