d*i
2 楼
明显有问题啊，难道是被帅哥绑架了？
t*t
3 楼
I'm using ZymoResearch EZ DNA kit to convert genomic DNA. Unmethylated and
methylated DNA controls from Milipore were converted together with my
samples.
I use qPCR based method to check methylation. Basically, if it's methylated,
the Tm will be higher that unmethylated DNA due to less C->T conversion.
My experiments were fine until recently. I always found all my samples,
control DNA (methy+unmethy) had the same Tm, which is even lower than (~2
degrees) supposed unmethylated DNA Tm. It looked that everything was "over-
converted".
Can anyone give me some suggestions?
methylated DNA controls from Milipore were converted together with my
samples.
I use qPCR based method to check methylation. Basically, if it's methylated,
the Tm will be higher that unmethylated DNA due to less C->T conversion.
My experiments were fine until recently. I always found all my samples,
control DNA (methy+unmethy) had the same Tm, which is even lower than (~2
degrees) supposed unmethylated DNA Tm. It looked that everything was "over-
converted".
Can anyone give me some suggestions?
a*n
4 楼
如果你父母第一次入境，一般都会6个月的，3个月可能是因为来美国太过于频繁，
在美国的时间比在国内的时间长。当然要运气不好也没办法。
【在 S*********2 的大作中提到】
: 。。后来听说一般只给三个月，是不是真的啊
: 我是F1学生
在美国的时间比在国内的时间长。当然要运气不好也没办法。
【在 S*********2 的大作中提到】
: 。。后来听说一般只给三个月，是不是真的啊
: 我是F1学生
w*i
5 楼
她给我发了俩包子，不过说不定是在和帅哥喝着小酒的时候给我马克的
【在 d******i 的大作中提到】
: 明显有问题啊，难道是被帅哥绑架了？
【在 d******i 的大作中提到】
: 明显有问题啊，难道是被帅哥绑架了？
s*s
6 楼
没做过tm，要么你seq以下看看？
methylated,
【在 t****t 的大作中提到】
: I'm using ZymoResearch EZ DNA kit to convert genomic DNA. Unmethylated and
: methylated DNA controls from Milipore were converted together with my
: samples.
: I use qPCR based method to check methylation. Basically, if it's methylated,
: the Tm will be higher that unmethylated DNA due to less C->T conversion.
: My experiments were fine until recently. I always found all my samples,
: control DNA (methy+unmethy) had the same Tm, which is even lower than (~2
: degrees) supposed unmethylated DNA Tm. It looked that everything was "over-
: converted".
: Can anyone give me some suggestions?
methylated,
【在 t****t 的大作中提到】
: I'm using ZymoResearch EZ DNA kit to convert genomic DNA. Unmethylated and
: methylated DNA controls from Milipore were converted together with my
: samples.
: I use qPCR based method to check methylation. Basically, if it's methylated,
: the Tm will be higher that unmethylated DNA due to less C->T conversion.
: My experiments were fine until recently. I always found all my samples,
: control DNA (methy+unmethy) had the same Tm, which is even lower than (~2
: degrees) supposed unmethylated DNA Tm. It looked that everything was "over-
: converted".
: Can anyone give me some suggestions?
S*2
7 楼
太感谢了。。
【在 a*****n 的大作中提到】
: 如果你父母第一次入境，一般都会6个月的，3个月可能是因为来美国太过于频繁，
: 在美国的时间比在国内的时间长。当然要运气不好也没办法。
【在 a*****n 的大作中提到】
: 如果你父母第一次入境，一般都会6个月的，3个月可能是因为来美国太过于频繁，
: 在美国的时间比在国内的时间长。当然要运气不好也没办法。
d*e
8 楼
帅哥被我灌醉了
我要去忙了
【在 w***i 的大作中提到】
: 她给我发了俩包子，不过说不定是在和帅哥喝着小酒的时候给我马克的
我要去忙了
【在 w***i 的大作中提到】
: 她给我发了俩包子，不过说不定是在和帅哥喝着小酒的时候给我马克的
R*n
9 楼
you are talking about Methylation Specific-HRM, I assume. Althought you are
using qPCR system, you didn't use a beckman probe to identify the sequence
change. So this is not a qPCR method.
ZymoResearch's kit should be fine. We used their kit a lot. There are some
commercial M or UM DNA standard which have been converted. You can use them
to test your primers.
How do you design your primers? flanking any CpG sites?
If you didn't change primer, I guess that you may have PCR product
contamination in your standard and DNA sample. This is a very possible
explanation.
using qPCR system, you didn't use a beckman probe to identify the sequence
change. So this is not a qPCR method.
ZymoResearch's kit should be fine. We used their kit a lot. There are some
commercial M or UM DNA standard which have been converted. You can use them
to test your primers.
How do you design your primers? flanking any CpG sites?
If you didn't change primer, I guess that you may have PCR product
contamination in your standard and DNA sample. This is a very possible
explanation.
J*i
10 楼
忙些什么
【在 d********e 的大作中提到】
: 帅哥被我灌醉了
: 我要去忙了
【在 d********e 的大作中提到】
: 帅哥被我灌醉了
: 我要去忙了
t*t
11 楼
Thanks. It's Methylation Specific-HRM. I'm studying the same CpG islands,
so primers are all the same.
Primers do flank CpG sites.My primers work.
Even I have PCR product contamination, how it is possible to get a Tm lower
than unmethylated control? I have PCR controls, which are already converted
standard DNA (methy and unmethy). They were fine with the right Tms.
Also, although I'm using the same reagents (kit, buffer,...), last week
there was once that my results were fine. Really got confused.
Is there any other recommended kit?
so primers are all the same.
Primers do flank CpG sites.My primers work.
Even I have PCR product contamination, how it is possible to get a Tm lower
than unmethylated control? I have PCR controls, which are already converted
standard DNA (methy and unmethy). They were fine with the right Tms.
Also, although I'm using the same reagents (kit, buffer,...), last week
there was once that my results were fine. Really got confused.
Is there any other recommended kit?
d*e
12 楼
你问的太多了
【在 J********i 的大作中提到】
: 忙些什么
【在 J********i 的大作中提到】
: 忙些什么
R*n
13 楼
If memory serves me correctly, kits usually use either beads or spin column
to capture the modified DNA strands.
Is your DNA come from cell or clinical sample? You can wash modified DNA
with TE buffer or your kit buffer a couples of more times to make sure
templates clean. Sometime I did have strange melting curves from clinical samples due to inpurities from biofluids. Or you can dilute your templates to see if there is any change.Even sub-nanogram concentration should work.
From your description, sounds like a primer dimmer or nonspecific PCR
product. I was using HRM kits from qiagen,roche and ABI. Qiagen works best.
Remeber HRM dye is high concentration salt.
Cheers,
Rinman
to capture the modified DNA strands.
Is your DNA come from cell or clinical sample? You can wash modified DNA
with TE buffer or your kit buffer a couples of more times to make sure
templates clean. Sometime I did have strange melting curves from clinical samples due to inpurities from biofluids. Or you can dilute your templates to see if there is any change.Even sub-nanogram concentration should work.
From your description, sounds like a primer dimmer or nonspecific PCR
product. I was using HRM kits from qiagen,roche and ABI. Qiagen works best.
Remeber HRM dye is high concentration salt.
Cheers,
Rinman
J*i
14 楼
不懂就要问
【在 d********e 的大作中提到】
: 你问的太多了
【在 d********e 的大作中提到】
: 你问的太多了
d*e
15 楼
自己琢磨
我睡觉去了
【在 J********i 的大作中提到】
: 不懂就要问
我睡觉去了
【在 J********i 的大作中提到】
: 不懂就要问
d*i
16 楼
嗯，问题大大的有啊
【在 d********e 的大作中提到】
: 自己琢磨
: 我睡觉去了
【在 d********e 的大作中提到】
: 自己琢磨
: 我睡觉去了
w*i
17 楼
烦妞儿要人跑了，你没有危机感？
【在 d******i 的大作中提到】
: 嗯，问题大大的有啊
【在 d******i 的大作中提到】
: 嗯，问题大大的有啊
r*r
18 楼
要人跑了，啥意思？
【在 w***i 的大作中提到】
: 烦妞儿要人跑了，你没有危机感？
【在 w***i 的大作中提到】
: 烦妞儿要人跑了，你没有危机感？
w*i
19 楼
跑人，呵呵，写反了
【在 r*******r 的大作中提到】
: 要人跑了，啥意思？
【在 r*******r 的大作中提到】
: 要人跑了，啥意思？
T*e
20 楼
怎么个跑法？有人抢亲？
【在 w***i 的大作中提到】
: 跑人，呵呵，写反了
【在 w***i 的大作中提到】
: 跑人，呵呵，写反了
w*i
21 楼
也可以跟人走啊
【在 T********e 的大作中提到】
: 怎么个跑法？有人抢亲？
【在 T********e 的大作中提到】
: 怎么个跑法？有人抢亲？
T*e
22 楼
哎呀，那我咋办？
【在 w***i 的大作中提到】
: 也可以跟人走啊
【在 w***i 的大作中提到】
: 也可以跟人走啊
O*y
23 楼
这下总算知道忙什么了
---小姨
【在 d********e 的大作中提到】
: 自己琢磨
: 我睡觉去了
---小姨
【在 d********e 的大作中提到】
: 自己琢磨
: 我睡觉去了
m*o
24 楼
你也可以学太后约会。。。
【在 T********e 的大作中提到】
: 哎呀，那我咋办？
d*i
25 楼
不要教坏小孩子
【在 m***o 的大作中提到】
:
: 你也可以学太后约会。。。
【在 m***o 的大作中提到】
:
: 你也可以学太后约会。。。
m*o
26 楼
太后肯定累坏了，让她休息吧。太后连给咱们发包子的力气也没有了。。。
【在 d******i 的大作中提到】
: 不要教坏小孩子
w*i
27 楼
我这不就是在问你吗？
【在 T********e 的大作中提到】
: 哎呀，那我咋办？
【在 T********e 的大作中提到】
: 哎呀，那我咋办？
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