problem with WGA amplification of my chip sample - (Sep/02/2008 )
Hi, I've been trying to amplify my chip samples with WGA kit from sigma. So far the positive control works perfectly. I get about 10ug from 10ng genomic DNA included in the kit. However, with same amount of chip samples I've only get 0.3-0.5ug. I searched the forum and it seemed that nobody has this problem before. It's so frustrating. Sigma technical support suggests that I can amplify for 20 cycles instead of 14 and they think iincreasing the cycle numbers to 20 will not introduce much more bias compared to 14 cycles. I'm wondering whether anybody has a similar experience before?
Thanks a lot. I 
appreciate your help.
Thanks a lot. I
appreciate your help.Sigma also told me I could use 20 cycles, but I found this was too much!
The Sigma WGA kit is so sensitive and prone to drama! How are you purifying your ChIP samples? We use a column (eluting in the tris buffer that comes with the kit). Perhaps something in your IP is inhibiting your reaction?
Also, how do you know you are amplifying 10ng of ChIP material? (easy for the input of course, but what about your IPs?).
We use 6 million cells/IP - elute in 13ul from the kit. We then amplify 1/4 and 1/2 of the IP sample. (We amplify two samples so as to make sure we are in the right phase of amplification - ie: our yield should double from 1/4 to 1/2).
Hope this helps
Clare
You should check if you are over sonicating your DNA. You should probably do no lower than an avg. size of 500 bp sonicated DNA.
Sigma also told me I could use 20 cycles, but I found this was too much!
The Sigma WGA kit is so sensitive and prone to drama! How are you purifying your ChIP samples? We use a column (eluting in the tris buffer that comes with the kit). Perhaps something in your IP is inhibiting your reaction?
Also, how do you know you are amplifying 10ng of ChIP material? (easy for the input of course, but what about your IPs?).
We use 6 million cells/IP - elute in 13ul from the kit. We then amplify 1/4 and 1/2 of the IP sample. (We amplify two samples so as to make sure we are in the right phase of amplification - ie: our yield should double from 1/4 to 1/2).
Hope this helps
Clare
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Thank you for your information. I also think the 20 cycles is too much. I use qiagen PCR purification column to purify CHIP sample and I use nanodrop to measure DNA concentration(only takes 1ul). I don't know what could be inhibiting my reaction in my sample, because I've already purified DNA with column. It's a good idea to amplify 1/4 and 1/2.
Thank you for the advice. Yes, from your standard, my DNA is oversonicated. I think the average size should be less than 400bp........ Increasing the DNA size would surely reduce the resolution, right? I will try to use less sonicated DNA to check what I can get. Thank you very much.