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”。