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http://www.mitbbs.com/article_t/Biology/32041171.html
【记录历史】NgAgo实验流程Addgene更新前后的比对
Reference：
原始发表的protocol
doi:10.1038/nbt.3547-s1, supplementary information
更新后的protocol
Addgene Han Lab added tips 2016年8月8日
https://www.addgene.org/78253/
under “RESOURCE INFORMATION” section “Supplemental Documents”
先看title
更新前
A general protocol of NgAgo/gDNA-mediated genome editing and examination (
T7E1 assay)
更新后
A general protocol of NgAgo/gDNA-mediated genome editing
再来看看实验流程包含的实验步骤
steps included in the protocol
更新之前的protocol包含以下7部分
1. Cell culture
2. Transfection
3. Genomic DNA extraction
4. PCR Amplification (a brief example)
5. T7EI reaction
6. PAGE analysis
7. Silver staining
更新之后的protocol包含以下4部分
1. Cell culture
2. Transfection
3. Genomic DNA extraction
Note:
看来韩春雨老师提到的“高超的实验技巧”应该在更新后的这4个部分里了。
下面慢慢来一条一条比对，做记录。
------
实验流程第一步：
1. Cell culture
更新前
293T cells are maintained in high-glucose DMEM (HyClone) supplemented with
10% FBS (HyClone) and penicillin/streptomycin at 37°C with 5% CO2
incubation. Cells are seeded to 24-well plate (Costar) with 60% confluence 8
hours before transfection. Thirty minutes before
transfection, cells are washed twice with PBS and medium is changed to high-
glucose DMEM medium containing 2% FBS.
更新后
293T cells are maintained in high-glucose DMEM (Gibco) supplemented with 10%
FBS (Gibco) and penicillin/streptomycin at 37°C with 5% CO2 incubation.
When cells reach their ≈60% confluence, perform transfection. Before
transfection, cells are changed to medium containing 2% FBS（heat
inactivated）. Since 293T cells are not firmly attached, be gentle and do
not disturb cells when changing medium.
更新后第一步的几个变化
1.特别注明293T cells
2.实验材料来源有变化 （Hyclone to Gibco）
3.transfection细节有变化
问题：那么在NBT原文中除了293T，也用到了几个其他类型的细胞，是不是每一种细胞
都有一个相应的不同的protocol？
---
实验流程第二步：
2. Transfection
更新前
2-1 NLS-NgAgo expressing plasmid is extracted with Wizard® Plus SV
Minipreps DNA Purification System (Promega), and is adjusted to 100 ng/μl
in 0.5x TE buffer (5 mM Tris-HCl, 0.5 mM EDTA，pH 8.0).
2-2 5’-phosphorylated ssDNA guides are dissolved to 100 ng/μl in 0.5x TE
buffer (PH 8.0). For each well of a 24-well plate, 200-250 ng NLS-NgAgo
plasmid and 100-300 ng guidesare diluted in 50 μl Opti-MEM (Gibco); 1.25 μ
l lipofectamine 2000 is diluted in 50 μl Opti-MEM. Incubate the DNA mix and
lipofectamine mix for 5 min.
2-3 Combine the DNA mix and lipofectamine mix with gentle pipetting and
incubate for 20 min. The DNA/lipo mixture is then added into each well of
cells.
*Since NgAgo follows “one-guide faithful” rule, i.e. guide can only be
loaded when NgAgo protein is in the process of expression, to improve the
efficiency of gDNA loading to NgAgo, multiple transfection of gDNA can be
conducted (e.g., 24 hours after the primary transfection)
*As stated below, cells will be harvested 48-60 hours after transfection. 90
% confluence of the cells on harvesting is ideal. Cell overplating
significantly weakens the efficacy of genome editing. Taking HDR as an
example, it occurs only during S and G2 phases.
更新后
2-1 NLS-NgAgo expressing plasmid is extracted with Wizard® Plus SV
Minipreps DNA Purification System (Promega), and is adjusted to 100 ng/μl
in water (pH 8.0, alkalization by NaOH).
2-2 5’ phosphorylated ssDNA guides are dissolved to 100 ng/μl in water (PH
8.0) For each well of a 24-well plate, 200-250 ng NLS-NgAgo expression
plasmid and 100-300 ng guides are diluted in 50 μl Opti-MEM (Gibco); 1.25
μl Lipofectamine® 2000 is diluted in 50 μl Opti-MEM. Incubate the
DNA mix and lipofectamine mix for 5 min.
2-3 Combine the DNA mix and lipofectamine mix with gentle pipetting and
incubate for 20 min. The DNA/lipo mixture is then added into each well of
cells.
*Since NgAgo follows “one-guide faithful” rule, i.e. guides can only be
loaded when NgAgo protein is in the process of expression, to improve the
efficiency of gDNA loading to NgAgo, sometimes multiple transfection of gDNA
helps (e.g. depending on the different kinetics of NgAgo expression in
different cells, a second transfection of gDNA can be performed 8, 12 or 24
hours after the primary transfection)
*As stated below, cells will be harvested 48-60 hours after transfection.
90% confluence of the cells on harvesting is ideal. Cell overplating
significantly weakens the efficacy of genome editing. Taking HDR as an
example, it occurs only during S and G2 phases.
更新后第二步的几个变化
1.NLS-NgAgo expressing plasmid 在提取后， “adjusted to 100 ng/μl in water
(pH 8.0, alkalization by NaOH)”， 原文是“adjusted to 100 ng/μl in 0.5x TE
buffer (5 mM，Tris-HCl, 0.5 mM EDTA，pH 8.0)”
2.5’ phosphorylated ssDNA guides 也从0.5xTE换成了water (PH 8.0)
3.ss gDNA二次转染时间有些变化， 更新后“multiple transfection of gDNA helps
(e.g. depending on the different kinetics of NgAgo expression in different
cells, a second transfection of gDNA can be performed 8, 12 or 24 hours
after the primary transfection)”，原文是“multiple transfection of gDNA can
be conducted (e.g., 24 hours after the primary transfection)”；更新前后都
包括了“24 hours”
问题：
1.更新后第一步的实验材料来源从HyClone改为Gibco， 原来在原文里protocol step 2
.2 藏着呢。。。。
2.更新后，从pH8.0的0.5xTE 变化到NaOH 调出来的pH8.0的水。那么不需要缓冲液的
pH8.0的水，pH稳定性有多好呢? 这个溶液是用于plasmid和 guides的， 都是DNA，如
果是要避免EDTA，为什么不直接用nuclease free water就好了呢？
3.两个protocol步骤2.2是一样的。在这步里面，我们计算一下稀释倍数
100ng/ul NLS-NgAgo expressing plasmid，取2-2.5ul （200-250ng），1-3ul guides
（100ng/ul，100-300ng），用50ul Opti-MEM (Gibco) 稀释后，再和lipofectamine
mix 混合； 原文用的是0.5xTE （5 mM Tris-HCl, 0.5 mM EDTA，pH 8.0），那么这步
稀释就是3-5.5ul DNA in 0.5xTE + 50ul Opti-MEM (Gibco)，总体积53-55.5ul，
EDTA的浓度在稀释后就大约是0.03-0.05mM了；后面还有一步和lipofectamine mix 混
合 （约2x稀释），那么原文中EDTA浓度在转染细胞前，就大概在0.015-0.025mM了。
更新后，就没有这个0.015-0.025mM EDTA了 ， 但是NBT原文的数据中，是用到了的。
4.两个流程里都提到了multiple transfection of gDNA，原文是建议24小时候再转染
一次 gDNA （24 hours after the primary transfection）， 更新后，特别注明二次
转染时间可以试试 8,12或者24小时
那么protocol更前新后，都包含了NBT原文中的“24小时”。
--------
实验流程第三步：
3.Genomic DNA extraction
更新前
3-1 48-60 hours after transfection, cells are harvested by trypsin digestion
. Four wells of cells are combined into a 1.5 ml EP tube.
3-2 For genomic DNA extraction, 500 μl of cell lysate buffer (50 mM Tris，
100 mM EDTA，0.5% SDS，pH 8) and 10 μl proteinase K (10 mg/ml）are added
into each tube and mixed gently and sufficiently. Bathe the tubes at 55℃
for 2 hours.
3-3 200 μl Tris-Phenol and 200 ul trichloromethane are added into each tube
and mixed gently and sufficiently. After incubation for 5 min, samples are
spun at 12,000 rpm for 15 min to separate aqueous phase from Phenol phase.
3-4 Carefully collect the aqueous phase into a clean EP tube.
3-5 Repeat the Steps 3-3 and 3-4 once and pool the aqueous phase.
3-6 Add 500 μl trichloromethane into the collected aqueous phase, mix
gently and sufficiently, stay for 5 min, and then spin the sample at 12,000
rpm for 15 min to separate aqueous phase from Phenol phase. Carefully remove
the aqueous phase into a clean EP tube.
3-7 Repeat the Steps 3-6 once.
3-8 Add 900 μl EtOH to the collected aqueous phase and incubate at -20°C
for
30 min.
3-9 Centrifuge the sample at 12,000 rpm for 10 min, and then the DNA
sediment is washed with 500 μl 75% EtOH thrice.
3-10 Air-dry the DNA sediment, add 50 μl 0.5 x TE and then adjust the
genome DNA to 100ng/μl for later use.
更新后
3-1 48-60 hours after transfection, cells are harvested by trypsin digestion
. Four wells of cells are combined into a 1.5 ml EP tube.
3-2 For genomic DNA extraction, 500 μl of cell lysate buffer (50 mM Tris，
100 mM EDTA，0.5% SDS，pH 8) and 10 μl proteinase K (10 mg/ml）are added
into each tube and mixed gently and sufficiently. Bathe the tubes at 55℃
for 2 hours.
3-3 200 μl Tris-Phenol and 200 ul trichloromethane are added into each tube
and mixed gently and sufficiently. After incubation for 5 min, samples are
spun at 12,000 rpm for 15 min to separate aqueous phase from Phenol phase.
3-4 Carefully collect the aqueous phase into a clean EP tube.
3-5 Repeat the Steps 3-3 and 3-4 once and pool the aqueous phase.
3-6 Add 500 μl trichloromethane into the collected aqueous phase, mix
gently and sufficiently, stay for 5 min, and then spin the sample at 12,000
rpm for 15 min to separate aqueous phase from Phenol phase. Carefully remove
the aqueous phase into a clean EP tube.
3-7 Repeat the Steps 3-6 once.
3-8 Add 900 μl EtOH to the collected aqueous phase and incubate at -20°C
for 30 min.
3-9 Centrifuge the sample at 12,000 rpm for 10 min, and then the DNA
sediment is washed with 500 μl 75% EtOH thrice.
3-10 Air-dry the DNA sediment, add 50 μl 0.5 x TE and then adjust the
genome DNA to 100 ng/μl for later use.
更新前后第三步基本没变化
-------
更新后的Note部分：
（NBT 原文里没有）
Note:
1.NgAgo/gDNA system is extremely sensitive to contamination of intracellular
bacteria (including mycoplasma) which are widespread and leave no visible
signs of presence. Carefully excluding the presence of intracellular
bacteria before performing experiments.
编者注：不能有细菌污染
2.Because Agos need magnesium, avoid EDTA when detaching and seeding cells
into the plates for transfection. Alternatively, supplementing Mg2+ to 5 mM
(may need optimization to your cell type).
编者注：avoid EDTA，optimize Mg2+
3.Avoid using Lipofectamine® 3000 since the supplement P3000 interferes
with ssDNA transfection. Lipofectamine® 2000 is a good choice. Other
transfection methods such as electroporation are yet to be tested.
编者注： 不要用 Lipofectamine® 3000；看来Lipofectamine® 2000是个不
错的选择
（ThermoFisher可要注意了，你们的Lipofectamine® 3000可能有问题！）
4. Ideally, 5' phosporylation of ssDNA guide by using T4 PNK (Biolab):
T4 PNK 2 μl
T4 ligase buffer (containing ATP): 12 μl
1OD ssDNA (about 33 μg) in H2O: 100 μl
(alternatively) additional ATP (25 mM) 2 μl
Add H2O to a final volume of 120 μl
Incubate at 37℃ overnight
After 5' phosphorylation, the resultant ssDNA needs no purification, dilute
by water (PH8) to 300 μl with a final concentration of 10 nM or 100 ng/μl)
编者注：
如果可以做到，要试试 5' phosporylation of ssDNA guide by using T4 PNK (
Biolab)
还别忘了最后要用PH8的水！
问题：鉴于前面有HyClone和Gibco的改变，如果不用“Biolab”的T4 PNK会有影响吗？
-----
问题总结
1.实验材料来源（Hyclone vs Gibco的培养基，以及原文没有特别提到的“Biolab”T4
PNK）会影响可重复性吗？
HyClone以及其他 T4 PNK 供应商，你们是不是要想一想自己的问题在哪里了？
还有，ThermoFisher可要注意了，你们的Lipofectamine® 3000可能有问题！
2.原文用于plasmid和guides （DNA）的0.5xTE，更新后 改成NaOH调出来的pH8.0的水。
先不谈怎么来用NaOH调出这个稳定的没有缓冲液的pH8.0的水，如果原因是由于NgAgo对
于EDTA这么的敏感，那么原文中用到的大约0.015-0.025mM的EDTA，是怎么做出的NBT文
章中的结果的？
3.关于细菌污染问题。从新闻报道和照片上看，韩春雨老师他们做实验是经常不戴手套
的，看来他们这样做细菌污染机率会减少。所以这些重复NgAgo实验的人们，你们重复
NgAgo实验的时候，带手套了吗？你们的实验材料有没有被细菌污染呢？
4.看来Mg2+的浓度，在不同的cell type也需要optimize
5.看来ss gDNA 二次转染的时间也需要优化。
"24 hours" 是protocol更新前后都有提到的，如果用了这个时间点来二次转染gDNA，
也做不出来，那就需要花不少时间和精力再去optimize了，如果真的可行的话。
6. guide DNA 的 5’ 磷酸化 在条件允许的情况下，也需要实验人员自己来优化
IDT 以及其他的各个引物提供商们， 你们也要注意了！
你们的引物合成，5’ 磷酸化修饰，以及质检等流程，需要好好检查一下了。
鉴于综上所述的各个条件，那么原文中这个实验结果， 该是花了多大功夫整出来的呀！
"We also tested 47 guides targeting eight different genes. The efficiency of
genome editing was comparable for all guides (21.3−41.3%; Fig. 4b,
Supplementary Fig. 5 and Supplementary Table 1), and we did not observe
obvious preferences of NgAgo to sequences with specific properties. We also
examined the NgAgo-gDNA system in a variety of mammalian cell lines
including breast cancer cell line MCF-7, human myeloid cell line K562 and
human epithelial cell line HeLa. In all of these cells, NgAgo could
efficiently induce targeted double-strand breaks at the DYRK1A gene (Fig. 4c
)."
---------
编者注：
1.韩春雨实验室在NBT文章中发表的数据，是用更新前的实验流程做出来的。
至今为止（2016年9月1日），世界上除了韩春雨实验室，还未有任何实验室能用这个更
新前的流程，重复验证出NBT文章中NgAgo基因编辑的能力。
2. 韩春雨实验室在公布更新后的实验流程以后，至今还未公布用此流程，任何成功重
复验证的结果。也没有任何一家其他实验室，能够公布用此流程而成功重复验证的结果。
因此：
- 在没有应用韩春雨所提出的“高超的实验技巧”之前，至今，世界上只有韩春雨一家
实验室，可以做出NgAgo基因编辑的结果
- 在韩春雨提出了“高超的实验技巧”之后，至今，世界上没有任何一家实验室，包括
韩春雨实验室，能够用这些“高超的实验技巧”，做出NgAgo基因编辑的结果
转载声明
-在注明出处 （mitbbs biology 版） 之后，编者授权全文转载。
2016年9月1日
CoinByCoin
|
|
楼主: newchinabok
发表于 2016-9-8 15:12
