最近要用PCR检测好几万个样品(玉米叶片或种子),请问有没有方法可以不用提DNA就可以直接做出PCR的?
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最近要用PCR检测好几万个样品(玉米叶片或种子),请问有没有方法可以不用提DNA就可以直接做出PCR的?
maybe可以,现在叶片pcr也挺多.水稻可以,玉米也还好吧~~~ 你试一下呗~~~
用拟南芥叶片直接作PCR得方法,共享如下:
取叶片(4平方毫米左右)置于PCR管,加0.25mol/L NaOH 40ul,沸水浴30sec,取出离心管,再加40ul 0.25mol/L HCl 和 20ul 缓冲液(0.5mol/L Tris-Cl, pH8.0; 0.25% NP-40),沸水浴中煮2min,室温下12000g 离心1min, 弃上清,于每个PCR管中加入50ul PCR反应液,开始PCR.
水稻也是差不多的,先加NaOH 研磨 再加TRis-HCL 研磨 后导入离心管 离心.最后跑PCR时用的模板就是离完心的上清.
直接用叶片做PCR:
Part I(a): Leaf Prep for PCR
Materials
1.5 mL microfuge tubes (with caps that stay closed when boiled)
Microfuge tube pestles (that fit the tubes tightly & reach to the tube bottom)
Dry ice crushed powder fine
Boiling water
0.5 N NaOH
Buffer: 0.2 M Tris, pH 8, 1mM EDTA
Cut off leaf (size = O) and place into the bottom of a labeled 1.5 mL microfuge tube and place onto dry ice. Place the pestles on dry ice also.
To obtain a good DNA yield, make sure the leaf is really frozen before you try to grind it. You will hear the leaf break as you grind it.
Collect up to 10-20 tubes on dry ice, grind the frozen leaf with a plastic microfuge tube pestle until it is powder fine and replace the tube on dry ice. (Use a different pestle for each leaf prep.)
Add 10 ul of 0.5 N NaOH to the tissue and thaw at room temp. Flick the tube a couple of times to be sure that the leaf bits are resuspended. (Note: You can hold the tubes at room temp for ~20 mins at this point.)
Give the tubes a quick spin in a microcentrifuge (5 sec).
Put the tubes into boiling water for 30 seconds.
After removing the tubes from the boiling water, add 100 ul of the Tris/EDTA buffer.
The preps should be placed on ice if they are to be used immediately or stored at -20 for later use.
Before setting up for PCR, vortex or mix the preps well to resuspend the leaf bits. Use 1 ul of the preps in 25-50 ul PCR reactions. (Note: Going higher than 1/20 DNA/reaction volume ratio can inhibit the reaction. Also, it seems to help if some leaf matter is included in the 1 ul.)
用拟南芥叶片直接作PCR得方法,共享如下:
取叶片(4平方毫米左右)置于PCR管,加0.25mol/L NaOH 40ul,沸水浴30sec,取出离心管,再加40ul 0.25mol/L HCl 和 20ul 缓冲液(0.5mol/L Tris-Cl, pH8.0; 0.25% NP-40),沸水浴中煮2min,室温下12000g 离心1min, 弃上清,于每个PCR管中加入50ul PCR反应液,开始PCR.
水稻也是差不多的,先加NaOH 研磨 再加TRis-HCL 研磨 后导入离心管 离心.最后跑PCR时用的模板就是离完心的上清.
直接用叶片做PCR:
Part I(a): Leaf Prep for PCR
Materials
1.5 mL microfuge tubes (with caps that stay closed when boiled)
Microfuge tube pestles (that fit the tubes tightly & reach to the tube bottom)
Dry ice crushed powder fine
Boiling water
0.5 N NaOH
Buffer: 0.2 M Tris, pH 8, 1mM EDTA
Cut off leaf (size = O) and place into the bottom of a labeled 1.5 mL microfuge tube and place onto dry ice. Place the pestles on dry ice also.
To obtain a good DNA yield, make sure the leaf is really frozen before you try to grind it. You will hear the leaf break as you grind it.
Collect up to 10-20 tubes on dry ice, grind the frozen leaf with a plastic microfuge tube pestle until it is powder fine and replace the tube on dry ice. (Use a different pestle for each leaf prep.)
Add 10 ul of 0.5 N NaOH to the tissue and thaw at room temp. Flick the tube a couple of times to be sure that the leaf bits are resuspended. (Note: You can hold the tubes at room temp for ~20 mins at this point.)
Give the tubes a quick spin in a microcentrifuge (5 sec).
Put the tubes into boiling water for 30 seconds.
After removing the tubes from the boiling water, add 100 ul of the Tris/EDTA buffer.
The preps should be placed on ice if they are to be used immediately or stored at -20 for later use.
Before setting up for PCR, vortex or mix the preps well to resuspend the leaf bits. Use 1 ul of the preps in 25-50 ul PCR reactions. (Note: Going higher than 1/20 DNA/reaction volume ratio can inhibit the reaction. Also, it seems to help if some leaf matter is included in the 1 ul.)
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