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molgen's Content

There have been 167 items by molgen (Search limited from 23-October 19)



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#55906 How Much BrET?

Posted by molgen on 20 January 2010 - 02:09 AM in Molecular Biology

I usually use ~10ng of Et-Br for every ml of TAE.



#55905 cDNA concentration higher than original RNA

Posted by molgen on 20 January 2010 - 01:48 AM in Molecular Biology

Did you take into consideration when doing the calculation that cDNA is both double stranded and a heteroduplex?



#55802 Antibody stability if left out at RT over Xmas break?

Posted by molgen on 19 January 2010 - 01:39 AM in General Lab Techniques

I would toss them and get a new batch.
In my experience, you will have a problem explaining your findings. Even if they work, the accuracy and accountability of the AB are compromised.
Using them after all, in my mind, is just bad science.



#55801 How to ask the author for an E. coli strains used in his paper?

Posted by molgen on 19 January 2010 - 01:29 AM in General Lab Techniques

The e-mail of the one of the authors is listed in the article for just such cases.



#55800 culture medium sterilization

Posted by molgen on 19 January 2010 - 01:23 AM in General Lab Techniques

Do you mean that polysaccharide clogs the filter?
If so, try preparing it in a sterile manner. If you got the polysaccharide from sigma then it comes sterile. Open it in a laminar flow and use sterile water do dissolve it. The just add it into the DMEM.



#55146 CENTRIFUGE SPEED

Posted by molgen on 14 January 2010 - 02:59 AM in General Lab Techniques

First, you have the molecular weight. The heavier something is the less time it needs to pellet.
Second, you can substitute speed and duration. i.e. if your centrifuge can't get up to the right speed you can increase the duration in the same proportion.



#54697 software for plasmid drawing

Posted by molgen on 10 January 2010 - 04:52 AM in General Lab Techniques

You can use pDRAW32 or GENtle or plasmid processor.
All of wich are freeware



#53205 How to Interpret it? Plz Help Me...

Posted by molgen on 28 December 2009 - 02:13 AM in General Lab Techniques

In the DNA sequence, the G (~3940) changed to an A causing the amino acid to change from D to N (1270).
In the middle you can see two carriers and their sick sun.
On the bottom, because of the mutation (D) you get another band.



#52536 Restriction Enzymes

Posted by molgen on 22 December 2009 - 12:33 AM in Molecular Biology

I have many RE from various source and usually don't have a problem with mix and matching. The only time it really was an issue, what I ended up doing is looking at the buffer composition and after the first digestion I added only the missing components from the buffer of the next digestion.
Make up a stock solution of 10 fold of the missing components and add it to the reaction buffer.
Good luck



#52531 Plant RNA Isolation Aid

Posted by molgen on 21 December 2009 - 11:43 PM in Molecular Biology

In my experience, the plant RNA isolation kits only work on things like tomato leaf or Arabidopsis.
For woody parts the kits don't work manly because thy are rich with lignin and suberin.
The cloudiness you mention probably is one of them becoming unsalable and that's why they clog your filters.
I'm sad to say that most probably you will have to use a CTAB protocol preferably with PVP.



#52254 TES

Posted by molgen on 21 December 2009 - 12:17 AM in General Lab Techniques

If I recall correctly, the advantage of the TES compared to the other buffers is manly in its buffer pH range (6.5-8.0)



#52253 cempetent e.coli and transformation

Posted by molgen on 21 December 2009 - 12:09 AM in General Lab Techniques

Using RPM isn't a good idea.
Different rotors in the same RPM will give different RCF or g.
You might be using to much force or not enough force.



#52093 bicarbonate in cell culture: powder v. liquid?

Posted by molgen on 20 December 2009 - 01:38 AM in General Lab Techniques

The powder is not sterile.
Adding it can contaminate your medium.
I'd dissolve it first, sterilize it and then add it to the medium.



#48875 Fast and Furious: Multiplex PCR

Posted by molgen on 02 December 2009 - 12:22 AM in PCR, RT-PCR and Real-Time PCR

I'm sorry if I didn't make my self clear.

I cited an article and then asked if you did something different.
If nothing is different then your PI probably doesn’t want to spend time and money on it.
If there is a novel thing then try running it again by your PI with a better pitch.



#48867 SYBR Green and Ethidium Bromide in same gel

Posted by molgen on 01 December 2009 - 11:58 PM in General Lab Techniques

But please do report back if you do this and tell us how it went.



#48864 Interpreting a Plasmid Profile gel photo

Posted by molgen on 01 December 2009 - 11:40 PM in General Lab Techniques

so then will the supercoil one be the least bottom one, the linear the middle one, and the nicked the top one?


This image will explain it best



#48659 Fast and Furious: Multiplex PCR

Posted by molgen on 01 December 2009 - 02:13 AM in PCR, RT-PCR and Real-Time PCR

The main "faster" effect is because of the different Taq and buffer, is not shorter PCR cycle (10-15 cycle) that many had thought (even my PI). That's the reason I don't understand why she couldn't take the idea...


I return to my original question:
What is new in this multiplex pcr that hasn't been dun before?

If it's only the speed of the reaction then why should she spend time and money on it?



#48657 Realtime PCR machine

Posted by molgen on 01 December 2009 - 02:07 AM in PCR, RT-PCR and Real-Time PCR

I think it depends on the chromophores you want to use



#48656 Bacteria Lysis Reagent For RNA extraction

Posted by molgen on 01 December 2009 - 01:59 AM in Molecular Biology

Adrian,
Have you tried any commercially available kit and fond it didn't work?



#48654 Interpreting a Plasmid Profile gel photo

Posted by molgen on 01 December 2009 - 01:50 AM in General Lab Techniques

Most probably the bands are super coiled, coiled and relaxed forms of the plasmid



#48347 Increasing the number of products your PCR produces

Posted by molgen on 29 November 2009 - 07:20 AM in PCR, RT-PCR and Real-Time PCR

Though the best way, in my mind, to go about this is to aligning all of the transcripts and to pick primers for the conserved regions, I can't see what good it will do you.
I think that the better science is to evaluate them separately (3'UTR for instance) and then sum them up.



#48345 PCR without thermal cycler?

Posted by molgen on 29 November 2009 - 07:10 AM in PCR, RT-PCR and Real-Time PCR

Chimp,
Have you noticed that article is almost 40 years old???
Things have changed.
Stagnation is not a virtue!!!



#48343 Fast and Furious: Multiplex PCR

Posted by molgen on 29 November 2009 - 06:52 AM in PCR, RT-PCR and Real-Time PCR

I think that your PI wants to know what is new in this multiplex pcr that hasn't been dun before.
I recently came across this article that did the same sort of thing.

Doing the same just faster but less accurate is bad from the clinical side. Say you give a wrong answer to someone in sepsis, you can kill him. Better wait a bit more, and get a better answer.



#48292 0.5% SDS

Posted by molgen on 29 November 2009 - 02:09 AM in General Lab Techniques

And wear a mask when you weigh the powder



#48291 Storage for mammalian blood samples

Posted by molgen on 29 November 2009 - 02:03 AM in General Lab Techniques

If you want to store gDNA, the best plase is in the fridge. That way the ice wont shred it.
For cDNA -20șC.
For live cells use 10% DMSO, 10-90% FCS (depending on the protocol) and freeze in -70șC for up to a month or liquid N2 for longer.




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