c*****n
发帖数: 195 | 1 哪位可以推荐一下yeast plasmid miniprep可靠的protocol？
万分感谢 | i*****g
发帖数: 93 | 2 zymo 有kit， 很好用的
【在 c*****n 的大作中提到】
: 哪位可以推荐一下yeast plasmid miniprep可靠的protocol？
: 万分感谢
| L******s
发帖数: 2349 | 3 加glass beads后votex，然后用e coli的miniprep kit就行
【在 c*****n 的大作中提到】
: 哪位可以推荐一下yeast plasmid miniprep可靠的protocol？
: 万分感谢
| w**t
发帖数: 52 | 4 We use the following protocol to prepare total DNA:
Zymolase/Sorbital/Tris/EDTA 1h @ 37C
SDS/EDTA/Tris 5min @ 65C
KAc 1h @ 4C
Top speed spin
isoprppanol/NH4Ac precipitate
70% EtOH wash
Dissolve in TE with RNase
The DNA prepared in this way could be transformed to E. coli to recover
plasmid or subjected for PCR for cloning. | c*****n
发帖数: 195 | 5 多谢各位的回复，用glass beads试过，可是分离出来的量太少， 只有50 ul, 10ug/ml
, 不够做sequencing的，看到很多人推荐zymo的kit，决定买来试一下
再次感谢各位的指点！ | f**u
发帖数: 346 | 6 那个beads得protocol分出来的质粒不是给你测序用的，一不够多二不够纯。
那个是让你用来转E. coli或者PCR的。
如果需要纯的质粒可以先转E. coli然后再抽出来。
ml
【在 c*****n 的大作中提到】
: 多谢各位的回复，用glass beads试过，可是分离出来的量太少， 只有50 ul, 10ug/ml
: , 不够做sequencing的，看到很多人推荐zymo的kit，决定买来试一下
: 再次感谢各位的指点！
| h**********r
发帖数: 671 | 7 以前做Neurospora的knockout的时候，看到过yeast方面的protocol。感觉他们挺严谨
的。但我不保证work。摘自：
http://www.dartmouth.edu/~neurosporagenome/Projects_files/Proje
Prepare yeast DNA with the Gentra yeast DNA kit:
(the following is slightly modified from their protocol:
http://www.gentra.com/pdf/01160.pdf)
the solutions are available from Gentra as Puregene kit D-6000A or
separately; if you have their Puregene solutions for Neurospora DNA preps,
you only need to order the Cell Suspension Solution (D-6001) and Lytic
Enzyme Solution (D-6002)
PUREGENE DNA Purification Kit
DNA Purification From 1 ml Yeast Culture Medium
Cell Lysis
1. Add 1 ml cell suspension (e.g., overnight culture) to a 1.5 ml tube on
ice.
2. Centrifuge at 13,000-16,000 x g for 5 seconds to pellet cells and remove
supernatant.
3. Add 300 μl Cell Suspension Solution to cell pellet and gently pipet up
and down
until cells are suspended.
4. Add 1.5 μl Lytic Enzyme Solution and invert tube 25 times to mix. [On
the robot we are using Zymolyase; details available on request: knockouts@
dartmouth.edu]
5. Incubate at 37°C for 30 minutes to digest cell walls. Invert sample
occasionally during
the incubation.
6. Centrifuge at 13,000-16,000 x g for 1 minute to pellet the cells. Remove
supernatant.
7. Add 300 μl Cell Lysis Solution to the cell pellet and gently pipet up
and down to lyse
the cells.
Protein Precipitation
1. Add 100 μl Protein Precipitation Solution to the cell lysate.
2. Vortex vigorously at high speed for 20 seconds to mix the Protein
Precipitation
Solution uniformly with the cell lysate.
3. Centrifuge at 13,000-16,000 x g for 3 minutes. The precipitated proteins
will form a
tight white pellet. If the protein pellet is not visible, repeat Step 2
followed by
incubation on ice for 5 minutes, then repeat Step 3.
[N.B. this is the point in the robot protocol at which we take the
supernatant and purify DNA from it with Agencourt’s CleanSEQ magnetic beads]
DNA Precipitation
1. Pour the supernatant containing the DNA (leaving behind the precipitated
protein
pellet) into a 1.5 ml microfuge tube containing 300 μl 100% Isopropanol
2. Mix the sample by inverting gently 50 times.
3. Centrifuge at 13,000-16,000 x g for 1 minute; the DNA might be visible as
a small
white pellet but don’t worry if you don’t see a pellet.
4. Pour off supernatant and drain tube briefly on clean absorbent paper. Add
300 μl
70% Ethanol and invert tube several times to wash the DNA pellet.
5. Centrifuge at 13,000-16,000 x g for 1 minute. Carefully pour off the
ethanol.
6. Invert and drain the tube on clean absorbent paper and allow to air dry
10-15
minutes.
DNA Hydration
1. Add 50 μl DNA Hydration Solution (50 μl will give a concentration in
the range of 100 to 200 ng/μl). (we do not do the recommended Rnase
treatment at this stage)
2. Rehydrate DNA by incubating sample 1 hour at 65ºC and/or overnight
at room
temperature. If possible, tap tube periodically to aid in dispersing the DNA.
3. Store DNA at 4°C. For long-term storage, store at -20°C or -80°C. | h**********r
发帖数: 671 | 8 同上另外一种方法。
Yeast “smash-and-grab” DNA prep:
• pipette 1-2 ml YPD onto transformation plate; scrape colonies off
with the end of a glass slide and pipette into a microfuge tube
• spin down 15 sec; pipette off sup; if cell pellet is more than 50-75
μl, remove excess and discard
• to cell pellet add 0.2 ml lysis buffer, 0.2 ml phenol/CHCl3 and 0.3
g 0.45-0.5 mm glass beads* (I use calibrated scoop made from cut microfuge
tube pierced with syringe needle) – seal carefully because beads can cause
sealing problem at rim
• vortex 1-2 min; wear gloves!
• spin 10 minutes; remove 100 μl supernatant to fresh tube (don’t go
too close to interface)
• to supernatant, add NaOAc to 0.3 M and 2.5 vol ethanol
• spin 3-5 min; discard supernatant; rinse pellet with 70% EtOH and
spin again
• air dry and resuspend pellet in 50 μl 1xTE
..there will be some background colonies due to end-joining or uncut vector
but it is not a problem
..if you want to purify the new KO plasmids, or if your PCR result is poor,
electroporate 1 μl of this DNA prep into E. coli and pick 6 or so to
analyze (chemical transformation didn’t work for me)
smash-and-grab lysis buffer:
2% Triton X-100
1% SDS
100 mM NaCl
10 mM Tris pH 8.0
1 mM EDTA
[based on M.D. Rose, F. Winston, and P. Hieter (1990) Methods in Yeast
Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, New York.]
*0.5 mm glass beads: the cheapest source I know of is Biospec http://www.biospec.com/Beads.htm Cat. No. 11079105 454g | h**********r
发帖数: 671 | 9 我在实验室用的Thermo，貌似work的不是很好。
Yeast DNA Extraction
(The protocol was modified by housepainter on December 28, 2009 according to
manufacture’s instructions)
Yeast DNA Extraction Kit, Thermo Number Description: 78870
Storage: Store at 4°C; if precipitant has formed, gently warm DNA Releasing
Reagents A and B at 37°C for 1-5 minutes.
Kit Contents:
Y-PER Reagent, 25 ml
DNA Releasing Reagent A, 20 ml
DNA Releasing Reagent B, 20 ml
Protein Removal Reagent, 10 ml
1. Pellet a 10 ml S. cerevisiae culture grown overnight, resuspend the cells
and transfer entire suspension to a 1.5 ml microcentrifuge tube. Pellet
cells by centrifugation at 3,000-5,000 × g for 5 minutes at room
temperature. Discard the supernatant. Typically this procedure will yield a
70-100 mg pellet.
2. Suspend cells in an appropriate amount of the Y-PER Reagent. Scale the
amount of Y-PER Reagent accordingly, maintaining a ratio of 8 μl/1 mg
pellet. Mix by gently vortexing or inverting the tube or pipetting up and
down until the mixture is homogenous. Once a homogenous mixture is
established, incubate at 65°C for 10 minutes.
3. Centrifuge at 3,000-5,000 × g for 5 minutes, discard supernatant, add
400 μl of DNA Releasing Reagent A, and 400 μl of DNA Releasing Reagent B
to the pellet for a total volume that should equal approximately 800 μl.
Mix to produce a homogenous mixture and incubate at 65°C for 10 minutes.
4. Add 200 μl of Protein Removal Reagent to mixture and invert several
times. Centrifuge at least 13,000 × g for 5 minutes and transfer
supernatant to a new 1.5 ml centrifuge tube.
5. Add 600 μl isopropyl alcohol to fill tube. Mix gently by inversion.
Precipitate genomic DNA by centrifuging the mixture at 13,000 × g for 10
minute.
6. Remove supernatant, being careful not to discard any of the pellet, which
is clear and hard to see. Add 1.5 ml 70% ethanol to the pellet, invert
several times and centrifuge at 13,000 × g for 1 minute to wash off any
residual salts or cellular debris clinging to the DNA or tube. Invert the
tube to dry any residual ethanol before proceeding to Step 7.
7. Resuspend in 50 μl TE buffer or sterile water. Pellet should solubilize
completely within 5 minutes. Flick the bottom of the tube carefully, or
pipette solution up and down. Wash the sides of the tubes until all the
genomic DNA is in solution.
Species and Strain Variations: This protocol was optimized with
. For DNA extraction from organisms that are difficult to lyse, allow the
samples to incubate for longer at Steps 2 and 3. Yield and RNA removal might
be significantly affected when using organisms other than S. cerevisiae. In
any case, no ribosomal RNA should be observed and small RNA species, if
present, may be removed by adding DNase-free RNase directly to the Y-PER
Reagent before Step 2. When stored at 4°C, RNase A is stable in the Y-PER
Reagent for over 6 months. To alter the DNA concentration, resuspend DNA in
differing amounts of TE or sterile water at Step 7.
Single Colony: When picking a single colony, use 20 μl of the Y-PER Reagent
, 16 μl of DNA Releasing Reagent A, 16 μl of DNA Releasing Reagent B, 8 μ
l of the Protein Removal Reagent, and 24 μl of isopropyl alcohol before
suspending the DNA in 5 μl of TE or sterile water. PCR amplification of
plasmid DNA is reproducible in this format. Use electroporation when
attempting to transform E. coli with DNA isolated from a single yeast colony
, because a high transformation efficiency is required. | h**********r
发帖数: 671 | 10 我以前在国内时候提细菌基因组用的方法，貌似对酵母也work,我以前就提过一次酵母
，确实work的很好，还记得gel上的照片贼亮贼亮的。那时候年轻啊！问过别人，说
lysozyme对yeast也能work.
Extraction of DNA from Bacteria and Yeasts
(This protocol was developed by Housepainter on February 09, 2010)
1.Inoculate 5 ml of medium with a single colony of transformed bacteria or
yeasts.
2.Pour 1.0 ml of the culture into a tube. Centrifuge at maximum speed for 30
seconds at room temperature. Remove the supernatant.
3.Resuspend the bacterial pellet in 1.0 ml 0.85% NaCl by vigorous vortexing.
4.Centrifuge at maximum speed for 30 seconds at room temperature. Remove the
supernatant.
5.Resuspend the bacterial pellet in 550 µl TE.
6.Add 17 µl of lysozyme (35 mg/ml) to each bacterial suspension. Store
the tube at 37°C for 30 minutes.
7.Add 3 µl of proteinase k (20 mg/ml) to each tube. Store the tube at
37°C for 30 minutes.
8.Add 30 µl of 10% SDS to each tube. Store the tube at 37°C for 30
minutes.
9.Add 100 µl of 5 M to each tube, mix thoroughly.
10.Add 80 µl of CTAB/NaCl solution to each tube. Mix and store the
tube at 65°C for 10 minutes.
11.Add an equal volume (about 0.7-0.8 ml) of phenol: chloroform. Mix and
then centrifuge the emulsion at 13,000 rpm for 10 minutes at room
temperature.
12.Transfer the aqueous upper layer to a fresh tube. Add an equal volume of
phenol: chloroform. Centrifuge at 13,000 rpm for 10 minutes.
13.Transfer the aqueous upper layer to a fresh tube. Add an equal volume of
chloroform. Centrifuge at 13,000 rpm for 10 minutes.
14.Transfer the aqueous upper layer to a fresh tube. Precipitate nucleic
acids from the supernatant by adding 0.6 volumes of isopropanol. Mix the
solution by vortexing and then allow the mixture to stand for 60 minutes at
room temperature.
15.Collect the precipitated nucleic acids by centrifugation at 13,000 rpm
for 10 minutes at 4°C.
16.Remove all of the supernatant. Add 1 ml of 70% ethanol to the pellet and
invert the closed tube several times.
17.Recover the DNA by centrifugation at 13,000 rpm for 10 minutes at 4°C.
18.Discard the supernatant. Store the open tube at room temperature until
the ethanol has evaporated and no fluid is visible in the tube (5-10 minutes
).
19.Dissolve the nucleic acids in 50 µl of TE (pH 8.0) containing 20 &
micro;g/ml DNase-free RNase A. Vortex the solution gently for a few seconds.
Store the DNA solution at -20°C. | | | h**********r
发帖数: 671 | 11 我记得我还有个专提yeast基因组的方法，找不着了。BTW，我不是主做yeast的。 | w**t
发帖数: 52 | 12 Yeast plasmid is low copy. I think even 2 micro origin plasmids (like pGADT7
or pGBKT7 for Y2H) present at around 100 copy per cell. You usually cannot
extract enough plasmid for the subsequent experiments such as sequencing.
Transform the total DNA to E. coli then extract the plasmid is much easier
then directly extract from yeast. | c*****n
发帖数: 195 | 13 多谢指点，第一次做，以为可以直接用来测序，这下明白了
【在 f**u 的大作中提到】
: 那个beads得protocol分出来的质粒不是给你测序用的，一不够多二不够纯。
: 那个是让你用来转E. coli或者PCR的。
: 如果需要纯的质粒可以先转E. coli然后再抽出来。
:
: ml
| a*****n
发帖数: 2835 | 14 如果是YTH的话，需要测序的话，可以做yeast colony PCR， 然后PCR产物加一种酶切
掉多余的引物，之后直接去测序，非常方便有效。等得到了序列信息再确定那些yeast
克隆需要提质粒转化细菌。这样工作量减小很多。
包子？
【在 c*****n 的大作中提到】
: 多谢指点，第一次做，以为可以直接用来测序，这下明白了
| c*****n
发帖数: 195 | 15 多谢多谢，
包子可以有，不过能告诉我怎么发吗？
yeast
【在 a*****n 的大作中提到】
: 如果是YTH的话，需要测序的话，可以做yeast colony PCR， 然后PCR产物加一种酶切
: 掉多余的引物，之后直接去测序，非常方便有效。等得到了序列信息再确定那些yeast
: 克隆需要提质粒转化细菌。这样工作量减小很多。
: 包子？
|
|
