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  2. → Viewing Profile: Posts: Wek

Wek

Member Since 30 Dec 2010
Offline Last Active May 10 2013 02:43 PM
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Posts I've Made

In Topic: Amount of RNA (ng) for good ct values.

17 February 2013 - 11:23 PM

View Postjerryshelly1, on 06 February 2013 - 11:57 AM, said:

What cDNA kit are you using?

Sorry for the late response.

Hmmm.. I don't really know the kit I am using to make cDNA. All the reagents I use were taken out of the package. I remember I use random hexamers and denature the RNA then add rnase, dntps, a buffer, and ssi (i think that's the name).

In Topic: Centrifugation speeds for cells.

06 February 2013 - 10:31 AM

I will try 100, 200, and 300 RCF for 5 mins and see if there is any significant loss of cells. My 3000 rpm equals to 800 RCF.
I have actually noticed the line smear on the wall of the tube when using compensation beads but haven't really noticed it when spinning down cells.

In Topic: Centrifugation speeds for cells.

05 February 2013 - 11:22 AM

View PostTabaluga, on 05 February 2013 - 11:12 AM, said:

Hm, I spin my cells down with 1300rpm for 3 min. Later during the FACS staining process I spin them down with 2400 rpm for 3 min, but be careful as this can harm the cells. Interestingly I also observe huge cell loss during the staining process although I never quantified it like you did... Do some of your cell stick to the wall of the reaction tube perhaps ?

Working with epithelia cells I use 3000 rpm for 3mins and working with HSCs the protocol calls for 1500 rpm for 5mins. I don't think my cells  are sticking to the wall of the reaction tube but I don't know for sure. I don't know if it is pipetting error, cell count error or the speed of spinning.

In Topic: Cell attachement occuring after 48 hours, not 24 hours.

02 January 2013 - 10:10 AM

View PostCurtis, on 02 January 2013 - 07:03 AM, said:

do you still have cells floating after 48 hours? or the ones that attached continued to divide?

After 48 hours most of the cells are attached, there are still a couple of cells floating but not as many as before.
After 72 hours, 99.9% of the cells are attached with barely any floaters.

In Topic: Quick question about making culture media.

26 December 2012 - 09:31 PM

How do you exactly make complete media the same way you would make a buffer (start with 60-80% base)? Wont you have to pour out the base media and measure it, increasing the chances of contamination? The media that we buy comes in 500ml bottles so we make 500ml of complete media every time.

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