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iamdhan's Content

There have been 11 items by iamdhan (Search limited from 13-October 18)


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#82812 abnormal nuclear morphology after treating shRNA

Posted by iamdhan on 08 August 2010 - 07:00 PM in Tissue and Cell Culture

Thanks for your comment, bob1! I will search for more information according to them. :)



#82514 olligo annealing for shRNA cloning

Posted by iamdhan on 05 August 2010 - 12:54 AM in siRNA, microRNA and RNAi

Hi Enthusiast,

In the protocol you mentioned above,

5ul Forward oligo
5ul Reverse oligo
5ul 10x NEB buffer2
35ul ddH2O

Is the "10X NEB buffer2" same as the reaction buffer they provide with restriction enzyme?

I have the exactly same problem now, and in my case, I am using 10x annealing buffer from Gendepot.

After several times of failure, I am not a fan of their annealing buffer. I am up to follow new protocol.



#82510 abnormal nuclear morphology after treating shRNA

Posted by iamdhan on 04 August 2010 - 11:45 PM in Tissue and Cell Culture

Hi,

After transfection with gene-of-my-interest-specific shRNA, there was a significant change in nuclear morphology.

It seems like it has something to do with maintaining normal nuclear structure, but I am not sure where to go from here with this.

I don't even know what to call this phenomenon in English. Therefore googling is not possible at the moment.

I know it is silly to show just one picture and ask other's opinion. Any short, rude, or half-minded reply might be a great help to me.

The confocal image is attached. Nucleus are colored in red and my gene is not in the frame cuz it was completely silenced.

I have checked control pool is perfectly normal.


What do you think? Why do nucleus have such a giant hole in them? It doesn't look like speckle.

Thanks!

Attached Thumbnails

  • data.jpg



#78819 the best loading control throughout cell cycle

Posted by iamdhan on 09 July 2010 - 01:52 AM in SDS-PAGE and Western Blotting

Hi,

Which protein could be a reliable loading control when your topic protein's expression level might be changeable?

Tubulin was what I used, but I've read from some paper that it is not stably synthesized during the cell cycle, so that's out of the option Iguess...

I specifically want to check if my protein plays a role in S or G2 phase.

As of now, Actin is my new loading control for sample from cytosol and Lammin for nuclear extracts.

Am I doing it right? Would I have some better choice?


I haven't had much experience in this feild, so my question could sound so stupid to you.

Any suggestion will help!

Thx.



#68108 chromatin isolation buffer..where to store?

Posted by iamdhan on 25 April 2010 - 06:45 PM in DNA Methylation and Epigenetics

Hi,

I am gonna make buffer according to this protocol: http://www.lamondlab...inisolation.pdf

It is for experiment called "Chromain Isolation by small-scale biochemical Fractionation"

Buffer A
10 mM HEPES, pH 7.9
10 mM KCl
1.5 mM MgCl2
0.34 M Sucrose
10 % Glycerol
1 mM DTT
Protease inhibitor cocktail

Buffer B
3 mM EDTA
0.2 mM EGTA
1 mM DTT
Protease inhibitor cocktail

I am wondering where I can keep those buffer; should I leave it at room temperature or in the fridge??

Thx!



#50336 contamination in establishing stable cell lines

Posted by iamdhan on 09 December 2009 - 02:16 AM in Tissue and Cell Culture

Hi,

I have a problem in establishing stable cell lines. After days of selecting for stable colonies, I picked on some of them, and now they are in 24-well dishes.

Before the wells get confluent, there is something flotting all over the wells. First I assumed that it is debris and ignored (cuz it seems exactly like debris!), but then, it starts to amplify. Believe me, despite of careful washing with PBS, these things won't dissapear and end up to get increased by size and number. Few days later I can even see them by naked eyes.

I threw them all out and started it from the scratch, but I am very depressed as it apprears again..

I attached an image of it. The stable cell of mine is in yellow circle and the one inside of the red circle is what I am talking about.

What do you think it is? Definitely I think it is something other than dead cells.

Attached Thumbnails

  • 20091209_ask.jpg



#36789 what is the point of serial dilution???

Posted by iamdhan on 17 September 2009 - 09:05 PM in Cell Biology

Hi all!

I am working on transfection in order to gain stable cells.

According to the method that I have, 24 hours after transfection of DNA to HeLa cell, I am supposed to perform serial dilution and wait for another 24 hours for cells to be stablized.

Here, I don't get why it needs serial dilution.

Thx a loooooooooot in advance!! :)



#35318 How long do you warm up media?

Posted by iamdhan on 03 September 2009 - 06:07 PM in Tissue and Cell Culture

Cool. <_< silly me, i worry too much.



#35223 How long do you warm up media?

Posted by iamdhan on 02 September 2009 - 05:54 PM in Tissue and Cell Culture

Any damage could be done to media if I warm it up too long?

Say....like half of a day before use?

I just forgot that I put it in 37'C water bath in the morning..

It was a one of those crazy days that i was mentally off..

Then 6 o'clock in the evening, same day, I just found a bottle of media, and was like "Hmm...that one seems familiar.........uh?......argg! it is mine!!!"

Would it be okay after extra hours of warming?

If not, why??

thx. :P



#35222 pellet became insoluble after boiling.

Posted by iamdhan on 02 September 2009 - 05:48 PM in SDS-PAGE and Western Blotting

Thanks guys!

She prepared new samples, and this time she made sure to boile them after appropriate times of pipetting. No aggregation. :P I didn't know how important this step is.



#34942 pellet became insoluble after boiling.

Posted by iamdhan on 31 August 2009 - 01:42 AM in SDS-PAGE and Western Blotting

Hello to all!

A colleague in my lab. boiled pellets(that she infected virus) with SDS in 100'C for 5 mins.

The thing is that she didn't mix the pellet with SDS by pipeting before she boiled it.

And to my surprise, it turned out to be a great mistake as the pellet somehow got in the shape and never is soluble at any rate; she tried everything to "destroy" the pellet like pipeting, vortexing, and flicking.

I don't know the full procedure of her experiment, so I might miss some obvious missing step, but nothing seems that wrong to deserve this unchangeable condition of pellet.

What do you think? How come this happens? Could possibly she make it soluble again?

Sorry for lack of detail and also for my broken english. :P


Thx!!




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