n*****s
发帖数: 8 | 1 I used Qiagen plasmid plus Maxiprep kit to purify my construct. And I was
asked to do a second precipitation after the elution step in order to
further remove the salt contamination. I use -20C isoproponal centrifuge at
room temp for 30 min, 15000xg, then wash with 70% ethanol, and resuspend
the DNA in EB buffer.
I did not see the precipitation at the bottom of the 2ml tube after the 30
min, a senior phd student told me that sometimes even one can not see the
precipitation one will still get some DNA. he suggested me to incubate the
DNA with EB buffer on a shaker (37ç, 300 rpm) for 3 hours... I never
heard this before but I still followed his words.
However, when I do the nanodrop to quantify my DNA, I got almost nothing...
I have to do all the maxiprep again (10 Maxiprep...), OMG!!!!!
what do you experts think will be the reason?
really need help!!!! |
h******y
发帖数: 1374 | 2 take it easy
at
【在 n*****s 的大作中提到】
: I used Qiagen plasmid plus Maxiprep kit to purify my construct. And I was
: asked to do a second precipitation after the elution step in order to
: further remove the salt contamination. I use -20C isoproponal centrifuge at
: room temp for 30 min, 15000xg, then wash with 70% ethanol, and resuspend
: the DNA in EB buffer.
: I did not see the precipitation at the bottom of the 2ml tube after the 30
: min, a senior phd student told me that sometimes even one can not see the
: precipitation one will still get some DNA. he suggested me to incubate the
: DNA with EB buffer on a shaker (37ç, 300 rpm) for 3 hours... I never
: heard this before but I still followed his words.
|
n*****s
发帖数: 8 | 3 淡定不了啊。。。
总得搞清楚是哪里出了问题啊。。。要不然下次还出问题怎么办。。。
老板好多钱就这么被我花掉了。。。
有经验的前辈给我分析一下吧！ |
m*****y
发帖数: 857 | 4 in most cases, you can see the precipitate from IPP centrifugation.
precipitate may be invisible from 70% EtOH precipitate though
at
【在 n*****s 的大作中提到】
: I used Qiagen plasmid plus Maxiprep kit to purify my construct. And I was
: asked to do a second precipitation after the elution step in order to
: further remove the salt contamination. I use -20C isoproponal centrifuge at
: room temp for 30 min, 15000xg, then wash with 70% ethanol, and resuspend
: the DNA in EB buffer.
: I did not see the precipitation at the bottom of the 2ml tube after the 30
: min, a senior phd student told me that sometimes even one can not see the
: precipitation one will still get some DNA. he suggested me to incubate the
: DNA with EB buffer on a shaker (37ç, 300 rpm) for 3 hours... I never
: heard this before but I still followed his words.
|
n*****s
发帖数: 8 | 5 是啊，以前做我能看到ipp导致的沉淀的，但是这一次。。。恨死了，好多钱好多时间
就这么没了，什么也没得到，哎。。。打起精神来，明天重新做！！ |
s******r
发帖数: 2876 | 6 The bacteria may be over grow,
try to pick up a new colony.
I used Qiagen plasmid plus Maxiprep kit to purify my construct. And I was
asked to do a second precipitation after the elution step in order to
further remove the salt contamination. I use -20C isoproponal centrifuge at
room temp for 30 min, 15000xg, then wash with 70% ethanol, and resuspend
the DNA in EB buffer.
I did not see the precipitation at the bottom of the 2ml tube after the 30
min, a senior phd student told me that sometimes even one can not see the
precipitation one will still get some DNA. he suggested me to incubate the
DNA with EB buffer on a shaker (37ç, 300 rpm) for 3 hours... I never
heard this before but I still followed his words.
However, when I do the nanodrop to quantify my DNA, I got almost nothing...
I have to do all the maxiprep again (10 Maxiprep...), OMG!!!!!
what do you experts think will be the reason?
really need help!!!!
【在 n*****s 的大作中提到】
: I used Qiagen plasmid plus Maxiprep kit to purify my construct. And I was
: asked to do a second precipitation after the elution step in order to
: further remove the salt contamination. I use -20C isoproponal centrifuge at
: room temp for 30 min, 15000xg, then wash with 70% ethanol, and resuspend
: the DNA in EB buffer.
: I did not see the precipitation at the bottom of the 2ml tube after the 30
: min, a senior phd student told me that sometimes even one can not see the
: precipitation one will still get some DNA. he suggested me to incubate the
: DNA with EB buffer on a shaker (37ç, 300 rpm) for 3 hours... I never
: heard this before but I still followed his words.
|
v*******a
发帖数: 759 | 7 “a second precipitation after the elution step in order to further remove
the salt contamination”
is it really necessary? |
n*****s
发帖数: 8 | 8 The bacteria may be over grow？？
可是我之前测了od值的，挺好的0.8ug每ul，在这个further precipitation之后就什么
dna都测不到了。。。 |
n*****s
发帖数: 8 | 9 这个construct之后要包装成病毒，所以需要remove the salt contamination，另一个
实验室在等着我的maxiprep，但是我现在都不敢用isopropanol的，我觉得问题可能出
在-20C 的 IPP上，本来我的volume就小，可以用room temp几分钟的。。。 |
s******r
发帖数: 2876 | 10 没太看明白你的过程，second precipitation？
max prep柱子洗脱之后，你是如何洗脱的？
你这时测了od？ 0.8ug/ul，
然后isopropanol沉淀？这是第一步沉淀？
The bacteria may be over grow？？
可是我之前测了od值的，挺好的0.8ug每ul，在这个further precipitation之后就什么
dna都测不到了。。。
【在 n*****s 的大作中提到】
: The bacteria may be over grow？？
: 可是我之前测了od值的，挺好的0.8ug每ul，在这个further precipitation之后就什么
: dna都测不到了。。。
|
|
|
f**u
发帖数: 346 | 11 呵呵，你这样能沉淀下来DNA才奇怪呢。DNA的酒精或者异丙醇沉淀是需要一定的盐浓度
的，常用的是0.3M NaAc。如果盐浓度不够是沉不下来的。你做Maxiprep的时候洗脱的
那一步是高盐洗脱，所以你第一遍沉淀不需要自己加盐，但是当你把沉淀溶解了以后二
次沉淀进一步纯化的时候一定要加盐才行。
另外说一句，你想把DNA里的盐完全去掉从化学上就是不可能的，磷酸骨架上的负电必
须要有正电的阳离子去中和，即使你用水去溶你的沉淀，或者用水从柱子上洗脱DNA，
都免不了有微量的盐。所以更可行的办法是找到一种和你的病毒包装实验compatible的
盐。由于Qiagen的elution buffer里的盐对于有些实验是不好的，所以有时候的确是需
要二次沉淀，不过沉淀前一定要自己调整盐浓度，一般来说加0.1体积的3M NaAc (PH 5
.2)，然后再用1体积异丙醇，或者三体积酒精沉淀。我的经验温度是不重要的。冰箱不
冰箱无所谓。
另外向Maxi这种很麻烦的，建议你离心以后把上清留着，这倒确认最后得到了DNA。另
外对于Maxi，绝对不可能看不到沉淀，你肯定能看到很大很大的沉淀才对，否则肯定是
哪里出问题了。对于Maxi，也真没有必要把二次沉淀的volume弄得太小，影响纯化效果
而且操作困难。
祝你后面的实验好运，感恩节快乐。 |
s******s
发帖数: 13035 | 12 1.没加盐
2.沉淀前没测OD
3.mini看不到沉淀有可能，maxi绝对不可能，那个三小时shaking完全搞笑
at
【在 n*****s 的大作中提到】
: I used Qiagen plasmid plus Maxiprep kit to purify my construct. And I was
: asked to do a second precipitation after the elution step in order to
: further remove the salt contamination. I use -20C isoproponal centrifuge at
: room temp for 30 min, 15000xg, then wash with 70% ethanol, and resuspend
: the DNA in EB buffer.
: I did not see the precipitation at the bottom of the 2ml tube after the 30
: min, a senior phd student told me that sometimes even one can not see the
: precipitation one will still get some DNA. he suggested me to incubate the
: DNA with EB buffer on a shaker (37ç, 300 rpm) for 3 hours... I never
: heard this before but I still followed his words.
|
w******a
发帖数: 1527 | 13 Qiagen maxiprep有时候真的很恶心。我后来换成zymo的了~~~~ |
c**y
发帖数: 127 | 14 zymo maxiprep只需要～40分钟？
这么快阿？
真是out了。。。
【在 w******a 的大作中提到】
: Qiagen maxiprep有时候真的很恶心。我后来换成zymo的了~~~~
|
M*****n
发帖数: 16729 | 15 you need more salt to precipitate DNA.
besides, never throw away the supernatant after maxiprep or precipitating
DNA, until you are sure you get what you want.
just my 2cents. |
n*****s
发帖数: 8 | 16 谢谢大家的回答，我虽然留着上清，但是我估计是不行了，要重新做了。。。
刚刚接触分子细胞3个月，发现一旦犯了错误，要浪费掉很多时间。。。真可怕。。。 |
w******a
发帖数: 1527 | 17 那个是很快的啦~~~~
【在 c**y 的大作中提到】
: zymo maxiprep只需要～40分钟？
: 这么快阿？
: 真是out了。。。
|
y***n
发帖数: 526 | 18 IPP沉完以后DNA pellet贴壁并不牢固，70% ethanol洗完以后会更松弛，所以倒液体的
时候要谨慎，不小心的话可能就把沉淀倒掉了 |
m*****z
发帖数: 1451 | 19 如果你是maxiprep的柱子elute之后加异丙醇沉淀没看到白色沉淀，十有八九是你的e
coli长老了。重新划板子挑新鲜菌落。
如果你确定在第二步沉淀前的确是有DNA的，那么你留的上清应该还是有用的，
Maxiprep这么多的DNA，加点NaCl就都能沉淀出来，50mM就够了。
我仍然很疑惑，maxiprep的产物最后都会用70%乙醇洗过，根本不存在高盐的问题啊，
不明白你这多一次的异丙醇沉淀意义何在
【在 n*****s 的大作中提到】
: 谢谢大家的回答，我虽然留着上清，但是我估计是不行了，要重新做了。。。
: 刚刚接触分子细胞3个月，发现一旦犯了错误，要浪费掉很多时间。。。真可怕。。。
|
