PCR troubleshooting - 未名空间MITBBS历史存档

国际科技财经博客移民网络热点娱乐民生时事公众号

Redian新闻

>未名空间

>Biology - 生物学

PCR troubleshooting

PCR troubleshooting# Biology - 生物学

b*r2012-10-17 07:10

1 楼

I am struggling for testing primers to genotype transgenic mice. I use test
1fg of vector as template mixed with 1ng of genomic DNA,
1. PCR always works when the template contains 1fg of vector but no genomic
DNA.
2.PCR always doesn't work when the template contains both 1fg of vector and
1ng of genomic DNA.
PCR conditions: 25 uL volume, pfu enzyme, 95 for 25",65 for 25",72 for 2'.
product size 1kb
Any solutions to fix this problem? Any PCR enzyme is able to PCR 1 fg of
target gene from genomic DNA?

g*n2012-10-17 07:10

2 楼

It seems that your genomic DNA samples contain something that inhibits the
enzyme. I would use Taq since fidelity is not a concern in genotyping.

b*r2012-10-17 07:10

3 楼

PCR本身连一两个copy，扩增的成功率都可以很高，见preimplantation diagnosis
你这个应该还是genomic DNA的问题？你怎么抽DNA的，测纯度没有，浓度怎么测的

b*r2012-10-17 07:10

4 楼

Thank for the message of you both.
"你怎么抽DNA的，测纯度没有，浓度怎么测的"
-->lysis buffer containing proteinase K digests mouse tail overnight at 65
degree. Phenol/chloroform isolates, ethanol precipitate. Dissolve in TE
buffer(pH7.5). 280/260 ratio=1.7-1.9, 浓度=0.2-0.5ng/uL
I tested 7 forward primers x 15 reverse primers. The totally primer pairs
are 105. All works well to PCR 1fg of plasmid which contain target gene, but
always don't work when template contains both 1fg of plasmid and 0.1ng of
mouse genomic DNA.
Any more suggestions?

【在 b****r 的大作中提到】

: PCR本身连一两个copy，扩增的成功率都可以很高，见preimplantation diagnosis
: 你这个应该还是genomic DNA的问题？你怎么抽DNA的，测纯度没有，浓度怎么测的

D*a2012-10-17 07:10

5 楼

你有含tg的老鼠尾巴么。
你酒精确定挥发干净了么？我们曾经有facility送过来的小鼠尾巴PCR不管用，就是因
为他们剪每只尾巴之前把剪子用酒精消毒，然后没干就剪尾巴了。但是我们知道了酒精
的问题以后，把管子打开晾很长时间用我们正常的protocol还是不能PCR。后来就让他
们必须晾干再剪或者不要用酒精消毒。
try
http://www.mitbbs.com/article_t/Biology/31638031.html

b*r2012-10-17 07:10

6 楼

Thank you for such a nice protocol.
"你酒精确定挥发干净了么？"-->good idea. I tried to get rid of ethanol
through dissolving purified genomic DNA in a tube with opened lid for 1 hour
at 45 degree of water bath. Not sure ethanol is remove completely.

【在 D*a 的大作中提到】

: 你有含tg的老鼠尾巴么。
: 你酒精确定挥发干净了么？我们曾经有facility送过来的小鼠尾巴PCR不管用，就是因
: 为他们剪每只尾巴之前把剪子用酒精消毒，然后没干就剪尾巴了。但是我们知道了酒精
: 的问题以后，把管子打开晾很长时间用我们正常的protocol还是不能PCR。后来就让他
: 们必须晾干再剪或者不要用酒精消毒。
: try
: http://www.mitbbs.com/article_t/Biology/31638031.html

D*a2012-10-17 07:10

7 楼

另外你说得不work是什么意思？
我们所有genotype都有内源control，也就是说无论老鼠是否tg，都有一条带确定PCR是
work的，tg老鼠再在tg大小位置上多一条带。
这样我们遇到酒精问题的时候才马上就确定是这一批样品都有问题。
另外你为啥要把vector跟wt genomic dna混起来，为啥不直接用转基因小鼠。

hour

【在 b******r 的大作中提到】

: Thank you for such a nice protocol.
: "你酒精确定挥发干净了么？"-->good idea. I tried to get rid of ethanol
: through dissolving purified genomic DNA in a tube with opened lid for 1 hour
: at 45 degree of water bath. Not sure ethanol is remove completely.

g*n2012-10-17 07:10

8 楼

It turns out as I suspected that something inhibited your enzyme. It is
phenol. You don't need to purify genomic DNA for genotyping. You can just
simply heat up your tail preps to 96oC for 10 min to kill proteinase and
take a bit from the preps for PCR.

b*r2012-10-17 07:10

9 楼

Sorry for this confusion.
It is too early for me to talk about transgenic mice. I am just during
electroporation of construct into ES cells. My Southern doesn't work for
genotyping. I have to use PCR to isolate positive ES cell clones. Then I
need get a primer pair for genotyping. Forward primer is from Neo, reverse
is from outside. What I did is knockin,not knockout.
Without purification of genomic DNA(just heat to kill proteinase K),PCR
doesn't work. After purification, still doesn't work.
"你为啥要把vector跟wt genomic dna混起来"-->I need pcr target recombinated
knockin gene, which is inserted in genomic DNA. This vector is test vector
which attempts to test primer pair's sensitivity. Apparently, most of
primers can't work if test vector is mix in genomic DNA.

【在 D*a 的大作中提到】

: 另外你说得不work是什么意思？
: 我们所有genotype都有内源control，也就是说无论老鼠是否tg，都有一条带确定PCR是
: work的，tg老鼠再在tg大小位置上多一条带。
: 这样我们遇到酒精问题的时候才马上就确定是这一批样品都有问题。
: 另外你为啥要把vector跟wt genomic dna混起来，为啥不直接用转基因小鼠。
:
: hour

D*a2012-10-17 07:10

10 楼

你试一下我那个digestion buffer吧，我们老鼠和细胞PCR都用那个，细胞我自己没做
过，貌似是要少加点，短一点时间。
另外一定要有internal positive control ！

【在 b******r 的大作中提到】

: Sorry for this confusion.
: It is too early for me to talk about transgenic mice. I am just during
: electroporation of construct into ES cells. My Southern doesn't work for
: genotyping. I have to use PCR to isolate positive ES cell clones. Then I
: need get a primer pair for genotyping. Forward primer is from Neo, reverse
: is from outside. What I did is knockin,not knockout.
: Without purification of genomic DNA(just heat to kill proteinase K),PCR
: doesn't work. After purification, still doesn't work.
: "你为啥要把vector跟wt genomic dna混起来"-->I need pcr target recombinated
: knockin gene, which is inserted in genomic DNA. This vector is test vector

b*r2012-10-17 07:10

11 楼

ok.Thanks a lot

b*r2012-10-17 07:10

12 楼

我觉得有个地方你没有搞清楚
plasmid几KB就有一个你要扩增的copy，gDNA要几十亿bp里才有一个你的copy
我的经验，一般的PCR条件，gDNA一般少于10ng就不好说能不能扩出来了。0.1ng？good
luck with that

but

【在 b******r 的大作中提到】

: Thank for the message of you both.
: "你怎么抽DNA的，测纯度没有，浓度怎么测的"
: -->lysis buffer containing proteinase K digests mouse tail overnight at 65
: degree. Phenol/chloroform isolates, ethanol precipitate. Dissolve in TE
: buffer(pH7.5). 280/260 ratio=1.7-1.9, 浓度=0.2-0.5ng/uL
: I tested 7 forward primers x 15 reverse primers. The totally primer pairs
: are 105. All works well to PCR 1fg of plasmid which contain target gene, but
: always don't work when template contains both 1fg of plasmid and 0.1ng of
: mouse genomic DNA.
: Any more suggestions?

b*s2012-10-17 07:10

13 楼

浓度=0.2-0.5ng/uL ==?
almost nothing in your sample..what's your nanodrop error threshold?
seems like you used too little gDNA template.
our genotyping PCR requires 0.1-1ug gDNA to start with, >100ng.

but

【在 b******r 的大作中提到】