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 |