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  2. → Viewing Profile: Posts: kblee

kblee

Member Since 29 Mar 2009
Offline Last Active Private
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Posts I've Made

In Topic: Can I go for ChP having frozen tissue

19 November 2012 - 12:11 AM

View Postbiznatch, on 05 June 2012 - 03:40 PM, said:

We do ChIP from frozen samples all the time.  The tissue is allowed to thaw slightly on ice, minced on ice with sharp dissecting scissors, resuspended in DMEM (no FBS) then passed (scraped) through a nylon cell strainer into a 50 mL tube with ~10 mL cold DMEM, everything is kept on ice.  Once the single cell suspension in DMEM is prepared formaldehyde is added to 1% final concentration and the protocol proceeds as normal.

Hi,
Can I ask why the tissue have to put in DMEM but not cold PBS or saline? And any difference between fresh tissue and snap frozen tissue? I will do NChIP for the both fresh and snap frozen tissue, any recommendation for me?

Thanks!

In Topic: CCRK (CDK20) protein expression and western blot

19 November 2012 - 12:04 AM

Can I know which antibody you are using to detect the CCRK? Since CCRK has 4 isoforms, do you know which antibody can detect the longest isoform? And which cells you use to overexpress CCRK? Thanks so much!

In Topic: CCRK (CDK20) protein expression and western blot

18 November 2012 - 11:42 PM

Thanks! Those three antibodies are all suitable for western blot.
Sign...

In Topic: CCRK (CDK20) protein expression and western blot

15 November 2012 - 07:26 PM

Blocking condition: 5% BSA or 5% milk, room temp for 1 hour
Primary antibody: using TBST or in blocking solution for antibody, dilution from 1:100 to 1:1000  4C O/N
Secondary antibody: using TBST or in blocking solution, dilution from 1:2000 to 1:5000 RT 2 hours
Protein amount: 30ug to 100ug

Yes, the sequence of my insert gene perfectly fine. In frame and without any mutation.

Thanks :D

In Topic: cloning from cDNA but got a much shorter PCR product

26 September 2012 - 12:19 AM

View Postbob1, on 25 September 2012 - 12:48 PM, said:

I take it that you are cloning from PCR - how are you doing this - linker ligation? TA cloning? tagged primers?

Have you optimised the PCR for a 1000 bp band before trying the cloning?

Yes I am cloning from PCR but my PCR product size doesn't match the size which search from UCSC.
The translated region of this gene is around 1000bp, but using the primer which carry restriction enzyme site to amplify cDNA, only 600bp and 400bp band size.
How can I amplify the gene from cDNA?

Thanks!

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