WGA problems - Input samples look terrible! - Any advice? (Dec/18/2007 )

Hi hi hi,

Just a quick question...

I am trying to get my WGA working for my Chip-chip. I need to amplify my DNA as I am using patient samples and therefore have limited material. Basically what I have been doing is taking all of my ChIP samples and amplifying - and they look great I still get the same fold-difference between my H3 and IgG samples. YAY! But.....I can never seem to get a good real time result after amplification of my input (I use GAPDH primers (positive control) and MYO-D (negative) to test via real time. Has anyone else had problems with amplifying input material? I have played around with difference starting concentrations of input DNA (1ng up to 60ng). I haven't played around with cycle numbers as I am using 20 cycles for my IP samples and hence want to do the same for my input.

Any advice would be greatly appreciated

Clare

Hi hi hi,

Just a quick question...

I am trying to get my WGA working for my Chip-chip. I need to amplify my DNA as I am using patient samples and therefore have limited material. Basically what I have been doing is taking all of my ChIP samples and amplifying - and they look great I still get the same fold-difference between my H3 and IgG samples. YAY! But.....I can never seem to get a good real time result after amplification of my input (I use GAPDH primers (positive control) and MYO-D (negative) to test via real time. Has anyone else had problems with amplifying input material? I have played around with difference starting concentrations of input DNA (1ng up to 60ng). I haven't played around with cycle numbers as I am using 20 cycles for my IP samples and hence want to do the same for my input.

Any advice would be greatly appreciated

Clare

Hi Clare,
what exactly do you mean with "Input samples look terrible"? Do you see a fold change after amplification? - then you should try a lower cycle number or less input. Sometimes it also might help to repeat decrsslinking.
[/quote]

It's all good now! I realised I was doing something wrong
But I still find that the real time PCR post amplification is never as nice for the input (but always nice for ChIP samples). By that I mean the standard curve is not as nice as before. I have just done my first hybridisation so am eager to see the results this afternoon

Hi everyone:

We are not doing Chip but MeDIP (methylated DNA immunoprecipitation), but we observe the same results as Clare after doing WGA on the MeDIP and Input DNA (we use the sigma kit and then purifying with microcon columns from milipore).

Standard curves on WGA Input look terrible.

Does anyone know what may be causing this?? Any advice??


Thanks.


Diana and Nina

I am seeing the same thing with qPCR. I ran exact same total DNA amount from Input pre-AMP and post-AMP. Get a big difference. I am wondering if people are purifying there WGA reaction with phenol chloroform or with qiaquick columns? If there is carry-over random primers from the WGA, it may interfere with the qPCR?

Hi Clare,
Sorry to bring back a really old thread, but I am having a similar problem with my input from my ChIP, sounds REALLY similar to your previous problem. You said you realized you were doing something wrong... can you tell me what the problem was, maybe i'm doing the same thing?

Thanks so much,

Angela

Hi Clare,
what exactly do you mean with "Input samples look terrible"? Do you see a fold change after amplification? - then you should try a lower cycle number or less input. Sometimes it also might help to repeat decrsslinking.


It's all good now! I realised I was doing something wrong
But I still find that the real time PCR post amplification is never as nice for the input (but always nice for ChIP samples). By that I mean the standard curve is not as nice as before. I have just done my first hybridisation so am eager to see the results this afternoon

Hi Angela,

Yeah, I was doing 20 cycles of WGA (as suggested by Sigma) but now I just do 14cycles and amplify 10ng and 20ng of my input
All good now!
Clare


[quote name='chipnewbie' date='Jun 4 2008, 10:57 PM' post='138411']
Hi Clare,
Sorry to bring back a really old thread, but I am having a similar problem with my input from my ChIP, sounds REALLY similar to your previous problem. You said you realized you were doing something wrong... can you tell me what the problem was, maybe i'm doing the same thing?

Thanks so much,

Angela