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Biology版 - genotyping digestion buffer
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请大家推荐一个提膜蛋白比较好的protocol或者kit, 做western用p32可以检测得到,h3才是危险?
相关话题的讨论汇总
话题: buffer话题: digestion话题: tail话题: genotyping话题: pcr
进入Biology版参与讨论
1 (共1页)
m******t
发帖数: 109
1
i want to know the digestion buffer for genotyping that doesnot purify
genomic DNA.i forgot it. let me know. thx.
D*a
发帖数: 6830
2
Quick Tail Prep
Collect 1 mg skin biopsy (= normal size tail or figure tip) in 0.5 ml
Eppendorf tube.
Add 100 microliter of quick tail digestion buffer (below).
Incubate @ 56 C several hours to overnight.
Vortex and incubate @ 99 C, 10 minutes.
Centrifuge @ max speed, 5 minutes.
(NB i don't vortex so i don't need to centifuge the tube, it works)
Use 1-2 microliter aliquot of supernatant for a 25-50 microliter PCR
reaction (or whatever you routinely do).
Transfer supernatant to a fresh tube for storage @ 4 C, if necessary.
(NB even without transfer the DNA is stable for long time, even 2 or 3
months, didn't count.cause i ususally use an old tube for positive control
until i finish it))
Quick Tail Digestion Buffer
50 mM KCl
10 mM Tris HCl, pH 9.0
0.1% Triton X-100
Add 0.4 mg/ml proteinase K just before use.
m******t
发帖数: 109
3
thx

【在 D*a 的大作中提到】
: Quick Tail Prep
: Collect 1 mg skin biopsy (= normal size tail or figure tip) in 0.5 ml
: Eppendorf tube.
: Add 100 microliter of quick tail digestion buffer (below).
: Incubate @ 56 C several hours to overnight.
: Vortex and incubate @ 99 C, 10 minutes.
: Centrifuge @ max speed, 5 minutes.
: (NB i don't vortex so i don't need to centifuge the tube, it works)
: Use 1-2 microliter aliquot of supernatant for a 25-50 microliter PCR
: reaction (or whatever you routinely do).

d****d
发帖数: 214
4
I use REDExtract-N-Amp Tissue PCR kit from Sigma.First put tail in
Extraction buffer @ RT for 10', then 95 C 3', then add Neutralization buffer
, then the sample is ready for PCR!

【在 m******t 的大作中提到】
: thx
m******t
发帖数: 109
5
i want to know the digestion buffer for genotyping that doesnot purify
genomic DNA.i forgot it. let me know. thx.
D*a
发帖数: 6830
6
Quick Tail Prep
Collect 1 mg skin biopsy (= normal size tail or figure tip) in 0.5 ml
Eppendorf tube.
Add 100 microliter of quick tail digestion buffer (below).
Incubate @ 56 C several hours to overnight.
Vortex and incubate @ 99 C, 10 minutes.
Centrifuge @ max speed, 5 minutes.
(NB i don't vortex so i don't need to centifuge the tube, it works)
Use 1-2 microliter aliquot of supernatant for a 25-50 microliter PCR
reaction (or whatever you routinely do).
Transfer supernatant to a fresh tube for storage @ 4 C, if necessary.
(NB even without transfer the DNA is stable for long time, even 2 or 3
months, didn't count.cause i ususally use an old tube for positive control
until i finish it))
Quick Tail Digestion Buffer
50 mM KCl
10 mM Tris HCl, pH 9.0
0.1% Triton X-100
Add 0.4 mg/ml proteinase K just before use.
m******t
发帖数: 109
7
thx

【在 D*a 的大作中提到】
: Quick Tail Prep
: Collect 1 mg skin biopsy (= normal size tail or figure tip) in 0.5 ml
: Eppendorf tube.
: Add 100 microliter of quick tail digestion buffer (below).
: Incubate @ 56 C several hours to overnight.
: Vortex and incubate @ 99 C, 10 minutes.
: Centrifuge @ max speed, 5 minutes.
: (NB i don't vortex so i don't need to centifuge the tube, it works)
: Use 1-2 microliter aliquot of supernatant for a 25-50 microliter PCR
: reaction (or whatever you routinely do).

d****d
发帖数: 214
8
I use REDExtract-N-Amp Tissue PCR kit from Sigma.First put tail in
Extraction buffer @ RT for 10', then 95 C 3', then add Neutralization buffer
, then the sample is ready for PCR!

【在 m******t 的大作中提到】
: thx
S******9
发帖数: 2837
9
50nm NaOH 500microliter 95-100度 煮30-45分钟
50 microliter Ph8 1M Tris-Hcl 中和
离心3分钟
取上清液做PCR
1 (共1页)
进入Biology版参与讨论
相关主题
p32可以检测得到,h3才是危险?请推荐一个做knockout小鼠genotyping的PCR kit
双酶切奇怪条带有人用sulfo-NHS-LC-biotin吗?
请推荐好的PIPETTE。Re: 谁从yeast里抽提过plasmid么?
realtime PCR仪器哪个好?请大家推荐一个提膜蛋白比较好的protocol或者kit, 做western用
求简单省钱的dna extraction protocol怎么GENOTYPE HOMOZYGOTE tg MICE
求一个好用的给genotyping PCR 的DNA isolation protocolwierd genotyping, and need help
核蛋白的Co-IP的 Protocolyeast miniprep
选eppendorf 还是beckman请教:BAC Maxi Prep的问题
相关话题的讨论汇总
话题: buffer话题: digestion话题: tail话题: genotyping话题: pcr