BG0056

EZgeneTM EndoFree Plasmid ezFlow
Maxiprep kit (BG0056)
Handbook
For research use only. Not intended for diagnostic testing.
www.abgent.com
Table of Contents
Introduction………………………………………………………….
2
Important Notes………………….…………………………………..
2
Storage and Stability………………………………………………...
3
Kit Contents………………………………………………...……….
4
Safety Information…………………………………………………..
5
Before Starting……………………………………………….……...
5
EndoFree Plasmid ezFlow Maxiprep Spin Protocol………………...
6
快速型无内毒素质粒大提…………………………………………
9
Purification of Low-Copy-Number Plasmid and Cosmid…………...
11
Trouble Shooting Guide..................................................…….……..
12
1
Introduction
Key to the plasmid purification kit is our proprietary DNA binding system that
allows the high efficient binding of DNA to our ezBindTM matrix while proteins
and other impurities are removed by wash buffer. Nucleic acids are easily eluted
with sterile water or Elution Buffer.
Unlike other kits in the markets, our patented plasmid purification kit has no
chaotropic salts in the buffer.The purified DNA is guanidine/anion exchange resin
residues free.
The EZgene™ endofree system uses a specially formulated buffer that extracts the
endotoxin from the bacterial lysate. The endotoxin level is less than 0.1 EU
(Endotoxin) per µg of plasmid DNA.
This kit is designed for fast and efficient purification of plasmid DNA from 150 to
400 mL of E. coli culture.The maxi column has a DNA binding capacity of 1200
µg. The purified endofree DNA is ready for downstream applications such as
transfection of endotoxin-sensitive cell lines, primary cultured cells or
microinjection.
Two endotoxin removal procedures are provided. Protocol A removes endotoxin
during the purification of plasmid DNA and Protocol B removes endotoxin after
the purification of plasmid DNA.
Important Notes
Plasmid Copy numbers
numbers:: The yield of plasmid DNA depends on the origin of the replication
and the size of the plasmid. The protocols are optimized for high copy number plasmid
purification. For low copy number plasmids, both the culture volume and the buffer volume
need to be scaled up 2 to 3 times. Reference Table 1 for the commonly used plasmids,
Table 1 Commomly used plasmids
5
Expected Yield
(µg per 200 mL)
12
P15A
10-12
25-40
pSuperCos
pMB1
10-20
30-50
pBR322
pMB1
15-20
35-50
pGEMR
Muted pMB1
300-400
350-450
pBluescriptR
ColE1
300-500
450-600
PUC
Muted pMB1
500-700
700-1,000
Plasmid
Origin
pSC101
pSC101
PACYC
Copy Numbers
2
Host Strains:
Strains:The strains used for propagating plasmid have significant influence
on yield. Host strains such as Top 10 and DH5a yield high-quality plasmid DNA.
endA+ strains such as JM101, JM110, HB101, TG1 and their derivatives, normally
have low plasmid yield due to either endogenous endonucleases or high
carbohydrates released during lysis. We recommend transform plasmid to an endAstrain if the yield is not satisfactory. Please reference Table 2 for the endA
information.
Table2 endA strains of E. Coli.
EndA- Strains of E. Coli
DH5α
DH1
DH21
JM106
JM109
SK2267
SRB
TOP10
DH10B
JM103
JM107
SK1590
MM294
Stbl2™
BJ5182
DH20
JM105
JM108
SK1592
Select96™
Stbl4™
XLO
XL1Blue
XL10Gold
EndA+ Strains of E. Coli
C600
HB101
JM110
TG1
JM101
JM83
All NM strains
RR1
ABLE® C
TB1
ABLE®
TKB1
HMS174
CJ236
KW251
DH12S
LE392
™
ES1301
M1061
All Y strains
K
P2392
PR700
Q358
BL21(DE3)
BL21(DE3)
pLysS
BMH 71-18
Optimal Cell Mass (OD600 x mL of Culture):
Culture):This procedure is designed for
isolating plasmid grown in standard LB medium (Luria Bertani) for 12 -16 hours to
a density of OD600 2.0 to 3.0. If rich medium such as TB or 2xYT are used, make
sure the cell density doesn’t exceed 3.0 (OD600). A high ratio of biomass over lysis
buffers result in low DNA yield and purity. The maxi column has an optimal
biomass of 450-550. For example, if the OD600 is 2.5, the optimal culture volume
should be 200 mL.
Culture Volume
Volume:: Use a flask or tube with a volume at 4 times the culture medium
to secure optimal condition for bacteria growth. Don’t exceed the maximum culture
volume suggested in the protocol. Incomplete lysis due to over amount of bacterial
culture results in lower yield and less purity.
Storage and Stability
Buffer A1 should be stored at 4°C once RNase A is added and Buffer ER should be
stored at 4°C. All other materials can be stored at room temperature (22-25°C). The
Guaranteed shelf life is 12 months from the date of purchase.
3
Kit Contents
alog #
Cat
Catalog
BG0055
BG0056
Preps
10
25
ezBindTM Columns
10
25
Buffer A1
110 mL
270 mL
Buffer ER
5.5 mL
14 mL
Buffer B1
110 mL
270 mL
Buffer D1
11 mL
28 mL
Buffer N3
40 mL
80 mL
Buffer RET
200 mL
2×250 mL
DNA Wash Buffer*
54 mL
2×54 mL
11 mg
(550 μL)
27 mg
(1.35 mL)
60 mL
135 mL
1
1
RNase A (20 mg/mL)
Endofree Elution Buffer
User Manual
*Add 216 mL (BG0055) or 2×216 mL (BG0056) 96-100% ethanol to each DNA
Wash Buffer bottle before use.
Safety Information
•
•
Buffer N3 contains acidic acid, wear gloves and protective eyewear when
handling.
Buffer N3and RET contains chaotropic salts, which may form reactive
compounds when combines with bleach. Do not add bleach or acidic solutions
directly to the preparation waste.
4
Before Starting
Alternative endotoxin removal procedures are provided. Protocol A removes
endotoxin during the purification of plasmid DNA and Protocol B removes
endotoxin after the purification of plasmid DNA.
Prepare all components and get all necessary materials ready by examining this
instruction booklet and become familiar with each step.
Important
�
RNase A: It is stable for more than half a year when stored at room temperature.Spin
down the RNase A vial briefly. Add the RNase A solution to Buffer A1 and mix well
before use. Store at 4°C.
�
Buffer ER should be stored at 4°C.
�
.It is critical to warm up the
Buffer B1 precipitates below room temperature
temperature.It
buffer at 50
50°°C to dissolve the precipitates before use.
�
Buffer N3 may form precipitates below 10
10°°C, warm up at 37
37°°C to dissolve the
precipitates before use
use..
�
Keep the cap tightly closed for Buffer B1 after use.
�
Make sure the availability of centrifuge, especially, after mixing the lysate with
ethanol, the sample needs to be processed immediately either by centrifugation.
�
In case the collection tube doesn’t fit in high-speed centrifuge rotor, use benchtop
centrifuge and spin at 2,500 x g with double centrifugation time. For example,
centrifuge at 2,500 x g for 10 min instead of 5,000 x g for 5 min.
�
�
Carry out all centrifugations at room temperature.
For certain reasons,Buffer KB are not involved in this kit from now on
Materials supplied by users
� 100% ethanol
� High speed centrifuge or vacuum manifold.
� 30 mL high speed centrifuge tubes and 50 mL tubes.
5
EZgeneTMEndoFree Plasmid ezFlow Max
Maxiiprep Spin
Protocol
Note:The Maxi column has a plasmid DNA capacity of more than 1.0 mg. To yield more
plasmid DNA, please use more bacterial culture. The following information is suitable for
high copy plasmid and the OD600 of culture is between 2.0-3.0. If the OD600 is lower than 2.0,
that will reduce the yield of the plasmid DNA. Please use more culture accordingly.
Table for the corresponding culture volume and solution volume
Culture
Volume
Buffer
A1/RNase
Buffer ER
Buffer B1
Buffer D1
Buffer N3
Buffer RET
100% ethanol
DNA Wash
Buffer
Endofree
Elution Buffer
< 200 mL
300 mL
400 mL
500 mL
Ratio
10 mL
15 mL
20 mL
25 mL
1V
0.5 mL
9 mL
1 mL
3 mL
0.7-1.0 V of
Supernatant
10 mL
0.75 mL
13.5 mL
1.5 mL
4.5 mL
0.7-1.0 V of
Supernatant
15 mL
1.0 mL
18 mL
2 mL
6 mL
0.7-1.0 V of
Supernatant
20 mL
1.25 mL
22.5 mL
2.5 mL
7.5 mL
0.7-1.0 V of
Supernatant
25 mL
0.05 V
0.9 V
0.1 V
0.3 V
0.7-1.0 V of
Supernatant
1V
10 mL
10 mL
10 mL
10 mL
10 mL
1.5-2.0 mL
1.5-2.0 mL
1.5-2.0 mL
1.5-2.0 mL
1.5-2.0 mL
Note: The volume of solution is supplied for 200 mL culture (OD600 between 2.0-3.0)
Please contact Abgent if you need more solutions.
•
150- 200 mL LB containing appropriate antibiotic with 100 µL fresh
Inoculate150starter culture. Grow at 37°C for 14-16 h with vigorous shaking.
Note
Note:The best way to prepare a starter culture: Inoculate a single colony from a freshly
grown selective plate into 1 mL LB medium containing the appropriate antibiotic and
grow at 37°C for 6-8 h with vigorous shaking (~250 rpm).
Note: Do not use a starter culture that has been stored at 4°C.
Note: Do not grow starter culture directly from glycerol stock.
Note:
Note:Do not use more than 200 mL culture or cell mass greater than 550.The buffer
volumes need to be scaled up if processing over 200 mL of culture.
2. Harvest the bacterial by centrifugation at 5,000 x g for 10 minutes at room
temperature. Pour off the supernatant and blot the inverted tube on paper towels
to remove residual medium completely.
6
Buffer A1 (Add RNase A to Buffer A1 beforeuse) and completely
3. Add 10 mL
mLB
resuspend bacterial pellet by vortexing or pipetting (Complete resuspension is
Bu
ffer ER into the suspended
critical for optimal yields).
yields).Then add 0.5 mL
mLBu
Buffer
bacterial culture. Mix well by inverting 5-10 times.
Note
Note: if room temperature is below 25°C, warm up the mix solution after adding
Buffer ER at 45°C for 5 min.
Buffer B1
4. Add 9 mL
mLB
B1, mix gently but thoroughly by inverting 5-10 times. If
necessary, continue inverting the tube until the solution becomes slightly clear.
Incubate at room temperature for 5 minutes to obtain a slightly clear lysate.
Buffer D1
Then add 1mL
1mLBuffer
D1, mix gently and incubating for another 5 minutes.
I.
Buffer N3
Add 3 mL
mLB
N3, mix immediately by sharp hand shaking for 5-10 times.
Transfer the sample to a high speed centrifuge tube and centrifuge at 12,000 x
g for 10 min at room temperature.
Note: It is critical to mix the solution well. If the mixture still appearsconglobated,
brownish or viscous, more mix is required to completely neutralize the solution.
Note: Syringe filter (Supplied in BG0057 or purchase separately from Abgent) could be
used to filtrate the lysate if high-speed centrifuge is not available.
II. Transfer
clear lysate to a clean 50 mL conical tube and add0.7-1.0
0.7-1.0
20 mL
mLclear
14 to 20m
Lof Buffer RET to 20 mLof
volume of Buffer RET (For example,14
20mL
clear lysate)
lysate),and 10 mL 100% ethanol
ethanol.Mix well by sharp hand shaking for 5
times. The mixture of the solution needs to be centrifuged through the DNA
column immediately.
Note: Increase the volume of Buffer RET could reduce endotoxin level in the plasmid
while the DNA yield will be affected by the Buffer RET. Different volume of Buffer
RET should bechosen according to the downstream application.
1. Immediately apply 20 mL of the solution into a DNA column with the
collection tube. Centrifuge at 5,000 x g for 2 min at room temperature.
Remove the column from the tube and discard the flow-through liquid. Process
the remaining solution
solution,, discard the flow-through liquid and put the column
back to the collection tube.
Note: If the 50 mL collection tube doesn’t match the rotor (for example, the lid of rotor
cannot close), the column with the collection can be centrifuged at a benchtop
centrifuge at > 2,500g for 5 min.
DNA Wash Buffer
2. Add 10 mL
mLDNA
Bufferinto the column, centrifuge at 2,500 x g for 1
7
min. Remove the column from the tube and discard the flow through. Reinsert
the column into the collection tube. Repeat step 8.
100% ethanol into the column, centrifuge at 2,500 x g for 1 min.
3. Add 3 mL
mL100%
Remove the column from the tube and discard the flow through. Reinsert the
column into the collection tub.
4. Centrifuge the column at 5,000 x g, with the lid open,
open,for 10 min to remove the
ethanol residues. Incubate at 65oC for 10 min will help to remove the ethonal
and increase the elution efficiency.
Note: Ethanol residues in the column will affect the elution of DNA. If the speed is less
than5,000 x g, centrifuge the column for 20 min and dry in the air to completely remove
the ethanol.
5.
Endofree
Transfer the column to a clean 50 mL tube and add 1.5-2.0 mL
mLEndofree
Elution Buffer
Bufferto the center of the column and incubate for 1 minute at room
temperature. Elute the DNA by centrifugation at >5,000 x g for 5 minutes.
6.
Reload the eluant into the center of the column and incubate for 1 minute.
Elute the DNA by centrifugation at>5,000 x g for 5 minutes.
Note:
Note:The DNA is ready for downstream applications such as cloning/subcloning, RFLP,
Library screening, in vitro translation, sequencing, transfection, and microinjection .
Note: Two elutions will increase DNA yield.
7.
Optional: Instead of step 12, for maximum the DNA yield, please add 4.0-5.0
Endofree Elution Buffer
mL
mLEndofree
Bufferto the center of the column and incubate for 5
minutes at room temperature. Elute the DNA by centrifugation at >5,000 x g
for 5 minutes. Reload the eluant twice to elute the plasmid DNA absorbed on
the membrane. This will give rise to maximum DNA yield.
Note: For DNA concentration, add 0.1 volume 3M Potassium Acetate or Sodium
acetate (pH 5.2) and 0.7 volume isopropanol. Centrifuge at top speed for 10 min.
Discard supernatant. Wash the DNA with 1 mL 70% ethanol, centrifuge for 5 min,
carefully decant. Air-dry the pellet for 20 minutes in a tissue culture hood. Resuspend
the DNA in Endofree Elution Buffer.
L)= OD260 nm x 50 x dilution factor.
DNA concentration (µg/m
g/mL
8
快速型去内毒素质粒大提试剂盒
详细资料请参阅英文资料
•
实验前准备
RNase A：室温下可稳定保存半年，使用前将提供的所有RNase A瞬时离心后加入Buffer A1, 使用后将
Buffer A1/RNase A置于4oC保存。
Buffer ER：保存于4oC。
Buffer B1：在低于室温时可能会沉淀，请于50oC左右水浴加热至沉淀完全溶解，溶液澄清，使用后保
证Buffer B1瓶盖旋紧。
Buffer N3：低于10oC会沉淀，请于37 oC左右水浴加热至沉淀完全溶解，溶液澄清。
准备100%的乙醇。
在室温下（22-25oC）进行所有离心操作。
•
注意事项
质粒拷贝数： 纯化中低拷贝的质粒时，请使用 2 倍体积的菌液，2 倍体积的 Buffer A1, ER, B1, D1,
N3, RET，相同体积的 DNA Wash Buffer 和 Endofree Elution Buffer。
转化菌：若为-70oC 甘油冻存的菌，请先涂布平板培养后，再重新挑选新的单个菌落进行培养。
切勿直接取冻存在 4oC 的菌进行培养。
柱结合能力：1 mg-1.5 mg
对富含内源核酸酶的宿主菌（ endA＋）如HB101, JM101, TG1等，需去内源核酸酶，请使用BG0069。
•
操作步骤
注：本试剂盒中柱子结合能力大于 1 mg，若实验中质粒得率较低，可以加大菌液用量。下表列出了处
理不同菌液应使用的溶液的量。下表的数据适用于 OD600 在 2.0-3.0 的高拷贝菌液。如果是低拷贝质粒
或者 OD 600<2.0，将会影响质粒的得率，请相应的增加菌液用量。
注：本试剂盒中提供的溶液量及操作步骤是根据 OD600 在 2.0-3.0 的高拷贝质粒的 200mL 菌液提供，如
需更多溶液，请联系 Abgent 公司。
Culture
Volume
Buffer
A1/RNase
Buffer ER
Buffer B1
Buffer D1
Buffer N3
Buffer RET
100% ethanol
DNA Wash
Buffer
Endofree
Elution Buffer
< 200 mL
300 mL
400 mL
500 mL
Ratio
10 mL
15 mL
20 mL
25 mL
1V
0.5 mL
9 mL
1 mL
3 mL
0.7-1.0 V of
Supernatant
10 mL
0.75 mL
13.5 mL
1.5 mL
4.5 mL
0.7-1.0 V of
Supernatant
15 mL
1.0 mL
18 mL
2 mL
6 mL
0.7-1.0 V of
Supernatant
20 mL
1.25 mL
22.5 mL
2.5 mL
7.5 mL
0.7-1.0 V of
Supernatant
25 mL
0.05 V
0.9 V
0.1 V
0.3 V
0.7-1.0 V of
Supernatant
1V
10 mL
10 mL
10 mL
10 mL
1.5-2.0 mL
1.5-2.0 mL
1.5-2.0 mL
1.5-2.0 mL
9
10 mL
1.5-2.0 mL
150-200 mL
1. 取100 µL新鲜的菌液接种到150-200
mL的LB培养基（含适量抗生素），37oC震荡培养14-16小时。
2. 室温下5,000 x g离心10分钟，收集菌体，并尽可能的吸去上清。
注：残留的液体培养基容易导致菌液裂解不充分，离心后沉淀较松，不能有效吸取上清。
注：本说明书中的操作程序适用于标准LB (Luria Bertani) 培养基培养12-16 小时后，OD600(细菌密度)
在2.0-3.0之间的菌液。若采用的是富集培养基，例如TB 或2×YT，请注意保证OD 600不超过3.0。
10 mL
Buffer A1
3. 加入10
mLBuffer
A1(确保已加入RNase A)，用移液器或涡流震荡确保细菌沉淀重新悬浮。 再向悬
0.5 mL
Buffer ER
浮的菌液中加入0.5
mL的Buffer
ER，反转5-10次，混合均匀。
注：不完全悬浮易导致菌体裂解不完全，从而使产量降低。
注：如果室温低于25°C，混合液在加入Buffer ER混匀后，置于45°C温育5min。
Buffer B1
4. 加入99 mL
mLBuffer
B1，轻轻地反转5-10 次以混合均匀，若有必要，请反复反转使溶液变为清澈无菌
团，在室温放置5 分钟。再加入11 mL Buffer D1
D1，轻轻地反转5-10 次以混合均匀，室温温育5分钟。
注：若溶液未清亮澄清，则表明菌体裂解不充分，应加大Buffer B1的用量或减少菌体量。
5. 加入33 mL Buffer N3
N3，用手甩3-5次使溶液充分混匀，此时出现白色絮状沉淀。将离心管转至高速离
心机，在室温下12,000 x g离心10分钟。
注：低温下RNase不工作，易有RNA污染。如果离心机转子较冷，将离心管在室温下温育 10分钟后
再离心。若无告速离心机，可用Syringe filter替代(在BG0057)中提供，也可单独购买)。
20 mL
6. 转移上清液20
mL(不超过20mL) 至新的50mL离心管中(若上清中仍有白色沉淀，可再次离心)，加入
7-1.0
Buffer RET
20 mL
14-20 mL Buffer RET)及10
10 mL
100% ethonal
0.
0.7-1.0
7-1.0倍体积的Buffer
RET(即每20
mL裂解液加入14-20
mL的100%
ethonal，
用手用力甩5次以混匀，溶液需马上离心过DNA柱。
注：使用1倍体积的Buffer RET(<1 EU/μg)，但是会稍微降低质粒的得率。请根据不同的实验需求选
择Buffer RET的使用体积。
20 mL
7. 立即转移20
mL裂解液/收集管至带收集管的DNA柱中，室温下5,000 x g离心2分钟，倒掉收集管中
的废液，将离心柱重新放回到收集管中。将剩余溶液转移至DNA柱中，室温下5,000 x g离心2分
钟，倒掉收集管中的废液，将离心柱重新放回到收集管中。重复直至所有的溶液通过DNA柱。
注：如果50 mL的收集管与离心机转子不符，可在台式离心机> 2,500 x g 离心5分钟。
10 mL
DNA Wash Buffer
8. 向离心柱中加入10
mLDNA
Buffer，室温下2,500 x g离心1分钟，倒掉收集管中的废液，将离
心柱重新放回到收集管中。重复步骤“88”。
100% ethonal
9. 向离心柱中加入33 mL
mL100%
ethonal，室温下2,500 x g离心1分钟，倒掉收集管中的废液，将离心柱
重新放回到收集管中。
10
10. 将离心柱放回高速离心机中，室温下5,000 x g开盖离心10分钟。离心后，将离心柱在65度烘箱中放
Endofree Elution Buffer 的洗脱效率。
置10分钟有助于彻底去除乙醇，提高Endofree
注：此步骤中开盖离心将会更有效的去除残留的乙醇，乙醇是否去除干净将会影响最后的洗脱效
率。若转速低于5,000 x g，需离心20分钟，并可放在空气中晾干。
1.5
Endofree Elution
11. 将离心柱转至一个新的50 mL离心管中，向DNA柱膜的正中加入1
.5--2.0 mL 的Endofree
Buffer
Buffer，室温放置1分钟，>5,000 x g离心5分钟，以洗脱质粒DNA。
12. 将50mL离心管中的洗脱液上柱再放置洗脱1分钟，>5,000 x g离心5分钟。两次洗脱将提高得率。
注：提取到的无内毒质粒DNA可用于转染内毒素敏感性细胞株，原代细胞及微注射。
13. 可 选： 不 用操 作第 12步 ，为 提高 DNA 的 得率 ，请 向柱 中央 加入 4.0-5.0 mL 的 Endofree Elution
Buffer
Buffer，温育5分钟，在>5,000 x g离心5分钟。再将洗脱液重新上柱重复洗脱两次。这将有利于洗脱
柱上吸附的所有的质粒DNA。
0.1
3M
pH5.2
0.7
注：得到的DNA需要浓缩，请加入0.1
0.1倍体积的3M
3M的醋酸钠（pH5.2
pH5.2）和0.7
0.7倍体积的异丙醇，室温
1mL
70%
下高速离心10分钟，底部可见白色的DNA沉淀，弃去上清，加入1mL
1mL70%
70%乙醇，高速离心5分钟，
Endofree Elution Buffer
弃去上清，空气干燥20分钟，加入Endofree
Buffer重新溶解质粒DNA。
Purification of Low-Copy-Number Plasmid and Cosmid
The yield of low copy number plasmid is normally around 0.1 – 1 µg /mL of
overnight culture. For isolating low copy number or medium copy number plasmid
DNA, use the following guideline:
1.
Culture volume: Use 2x volume of the high copy number culture. Use up
to 400 mL for the maxiprep.
2.
,Buffer ER, Buffer D1,
Use 2x volume of the Buffer A1, Buffer B1
B1,Buffer
Buffer N3
N3,, Buffer RET
RETand 100% ethanol
ethanol. Additional buffers can be
purchased from Abgent.
3.
ash Buffer and Endofree Elution Buffer
Use same volume of DNAW
DNAWash
Buffer.
11
Trouble Shooting Guide
Problems
Possible Reasons
Suggested Improvements
Low Yield
Poor Cell lysis.
� Resuspend pellet thoroughly by
votexing and pipetting prior
adding Buffer B1.
� Make fresh Buffer B1 if the cap
had not been closed tightly.
(Buffer B1: 0.2N NaOH and
1%SDS).
Low Yield
Bacterial culture
overgrown or not
fresh.
Grow bacterial 12-16 hours. Spin
down cultures and store the pellet at
-20°C. if the culture is not purified
the same day. Do not store culture at
4°C over night.
Low Yield
Low copy-number
plasmid.
No DNA
Plasmid lost in Host
E.coli
Increase culturevolume and the
volume of Buffer according to
instruction on page 8.
Prepare fresh culture.
Genomic DNA
contamination
Over-time incubation Do not vortex or mix aggressively
after adding Buffer
after adding buffer B1. Do not
B1.
incubate more than 5 minutes after
adding Buffer B1.
RNA contamination
RNase A not added
to Buffer A1.
Plasmid DNA floats
out of wells while
running in agarose
gel, DNA doesn’t
freeze or smell of
ethanol
Ethanol traces not
Make sure that no ethanol residual
completely removed remaining in the silicon membrane
from column.
beforeeluting the plasmid DNA. Recentrifuge again if necessary.
12
Add RNase A to Buffer A1.
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