Table of Contents Introduction... 2 Important Notes Storage and Stability... 4 Before Starting... 4 Kit Contents... 5 Safety Information.. 5 EZg

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1 EZgene TM Plasmid Miniprep Kit II (BG0005) Handbook For research use only. Not intended for diagnostic testing.

2 Table of Contents Introduction... 2 Important Notes Storage and Stability... 4 Before Starting... 4 Kit Contents... 5 Safety Information.. 5 EZgene TM Plasmid Miniprep II Spin Protocol EZgene TM Plasmid Miniprep II Spin/Vacuum Protocol Purification of Low-Copy-Number Plasmid/Cosmid. 9 质粒小提试剂盒 II 简明步骤. 10 Trouble Shooting Guide

3 Introduction Key to the kit is our proprietary DNA binding systems that allow the high efficient binding of DNA to our ezbind TM matrix while proteins and other contaminates are removed under certain optimal conditions. Nucleic acids are easily eluted with sterile water or Elution Buffer. This kit is designed for fast and efficient purification of plasmid DNA from 1 to 15 ml of E. coli culture. With the binding capacity of 80 µg, the yield obtained by Miniprep Kit II is higher than Miniprep Kit I. The yield from 1 ml culture is typically around 8 to 12 µg. The purified DNA is ready for downstream applications such as cloning/subcloning, RFLP, sequencing, and transfection of HEK293 cells. Important Notes Plasmi smid Copy 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 3 to 5 times. Please reference Table 1 for the commonly used plasmids, Table 1 commonly used plasmid and expected yield. Plasmid Origin Copy Numbers Expected Yield (µg per 1 ml) psc101 psc pacyc P15A psupercos pmb pbr322 pmb pgem R Muted pmb pbluescript R ColE puc Muted pmb Host 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 enda- strain if the yield is not satisfactory. 2

4 Table2 enda- strains of E. Coli. EndA- Strains of E. Coli DH5α DH1 DH21 JM106 JM109 SK2267 SRB XLO TOP10 DH10B JM103 JM107 SK1590 MM294 Stbl2 XL1- Blue BJ5182 DH20 JM105 JM108 SK1592 Select96 Stbl4 XL10- Gold EndA+ Strains of E. Coli C600 JM110 RR1 ABLE C CJ236 KW251 P2392 BL21(DE3) ABLE HB101 TG1 TB1 BL21(DE3) DH12S LE392 PR700 K plyss JM101 JM83 TKB1 HMS174 ES1301 M1061 Q358 BMH All NM strains All Y strains Optimal Cell Mass (OD600 x ml of Culture): This procedure is designed for isolating plasmid grown in standard LB medium (Luria Bertani) for hours to a density of OD to 3.0. If rich mediums such as TB or 2xYT are used, make sure the cell density doesn t exceed 3.0 (OD 600). A high ratio of biomass over lysis buffers result in low DNA yield and purity. The mini column has an optimal biomass of For example, if the OD600 is 3.0, the optimal culture volume should be ml. For over amount of cell numbers, either reduce the biomass or scale up the volumes of Buffer A1, B1 and N1. Culture Volume olume: Use a flask or tube 4 times bigger in volumn thanthe 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. 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

5 Before Starting Prepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each steps. Important RNase A: It is stable for more than half a year when stored at room temperature. Spin down RNase A vial briefly. Add the RNase A solution to buffer A1 and mix well before use. Store at 4 C. Add 60 ml (BG0004) or 60 ml (BG0006) or 96 ml (BG0005) % ethanol to each DNA Wash Buffer bottle before use. Buffer B1 precipitates below room temperature. It is critical to warm up the buffer at 50 C to dissolve the precipitates before use. Keep the cap tightly closed for Buffer B1 after use. Ensure the availability of centrifuge capable of 13,000 rpm. Carry out all centrifugations at room temperature. Materials supplied by users % ethanol 1.5 ml, 2.0 ml microcentrifuge tubes. 15 ml conical tubes. High speed microcentrifuge or Vacuum manifold. 4

6 Kit Contents Catalog# BG0004 BG0005 BG0006 Preps ezbind Columns Buffer A1 25 ml 125 ml 50 ml Buffer B1 25 ml 125 ml 50 ml Buffer N1 30 ml 135 ml 60 ml Buffer KB 30 ml 135 ml 60 ml DNA Wash Buffer* 15 ml 3 x 24 ml 2 x 15 ml Elution Buffer 15 ml 60 ml 30 ml RNase A (20 mg/ml) 2.5 mg (125 µl) 12.5 mg (625 µl) 5 mg (250 µl) User Manual *Add 60 ml (BG0004) or 60 ml (BG0006) or 96 ml (BG0005) % ethanol to each DNA Wash Buffer bottle before use. Safety Information Buffer N1 contains acidic acid, wear gloves and protective eyewear while handling. Buffer N1 and KB contains chaotropic salts, which may form reactive compounds when combines with bleach. Do not add bleach or acidic solutions directly to the preparation waste. 5

7 EZgene TM Plasmid Miniprep II Spin Protocol For 1-4 ml culture, reduce the volume of Buffer A1, B1, N1 to 250 µl, 250 µl and 350 µl, respectively. And use the same volume of DNA Wash Buffer and Elution Buffer. 1. Inoculate 5-12 ml LB containing appropriate antibiotic with a fresh colony. Incubate at 37 C for hours with vigorous shaking. Note: Prolonged incubation (> 16 hours) is not recommended since the E.coli starts to lyse and the plasmid yields may be reduced. Note: Do not grow the culture directly from the glycerol stock. Note: This protocol is optimized for E. coli strain cultured in LB medium. When using TB or 2xYT medium, special care needs to be taken to ensure the cell density doesn t exceed 3.0 (OD600). Buffers need to be scaled up proportionally if over amount of cultures are being processed. 2. Harvest bacterial culture by centrifugation for 1 min at 10,000 rpm. Pour off the supernatant and blot the inverted tube on a paper towel to remove residue medium. Remove the residue medium completely. Note: The culture can be centrifuged at 6,000 rpm in a 15 ml conical tube for 10 minutes if high speed centrifuge tubes are not available. Alternatively, the cultures can also be spin down in multiple 2.0 ml tubes. 3. Add 450 µl Buffer A1 (Add RNase A to Buffer A1 before use) and completely resuspend bacterial pellet by vortexing or pipetting. Note: Complete resuspension is critical for bacterial lysis and lysate neutralization. 4. Add 450 µl Buffer B1, mix gently by inverting 10 times (do not vortex) and incubate at room temperature for 5 minutes. If necessary, continue inverting the tube until the solution becomes slightly clear. Note: Do not incubate for more than 5 minutes. Note: Buffer B1 precipitates (cloudy look) below room temperature. Warm up Buffer B1 at 37 C to dissolve precipitation before use. 5. Add 550 µl Buffer N1, mix completely by inverting/shaking the vial for 5 times and sharp hand shaking for 3 times. Note: Incubating the lysate in ice for 1 min will improve the yield. Note: It is critical to mix the solution well. If the mixture still appears conglobated, brownish or viscous; more mixing is required to completely neutralize the solution. 6. Centrifuge the lysate at 13,000 rpm for 10 minutes at room temperature. Note: If the lysate doesn t appear clean, reverse the tube angle, centrifuge for 5 more 6

8 minutes and then transfer the clear lysate to DNA column. 7. Carefully transfer up to 700 µl clear lysate into a DNA column with a collection tube, avoid the precipitations, spin at 13,000 rpm for 1 minute, discard the flow-through and put the column back to the collection tube. Carefully transfer the remaining clear lysate to the column and centrifuge at 13,000 rpm for 1 minute at room temperature and discard the flow-through in the collection tube. Put the column back to the collection tube. 8. Add 500 µl Buffer KB into the spin column, centrifuge at 13,000 rpm for 1 minute. Remove the spin column from the tube and discard the flow-through. Put the column back to the collection tube. Note:This step is important to remove residual protein contaminations especially for enda+ strains and be highly recommended for high quality plasmid DNA.. 9. Add 650 µl DNA Wash Buffer (Add ethanol to DNA wash buffer before use) into the spin column, centrifuge at 13,000 rpm for 1 minute at room temperature. Remove the spin column from the tube and discard the flow-through. Repeat step 9 to improve the recovery. 10. Reinsert the spin column, with the lid open, into the collection tube and centrifuge for 5-10 minutes at 13,000 rpm. Note: Residual ethanol can be removed more efficiently with the column lid open. It is critical to remove residual ethanol completely. 11. Carefully transfer the spin column into a clean 1.5 ml tube and add µl ddh 20 or Elution Buffer into the center of the column and let it stand for 2 minute. Elute the DNA by centrifugation at 13,000 rpm for 1 minute. Reload the eluate into the column and centrifuge again to improve the recovery. Note:It is recommended to use Elution Buffer instead of ddh20. Note: The ph of Elution Buffer or ddh 2O will affect the plasmid DNA elution. If ddh 2O is applied, please make sure the ph is no less than 7.0 ( is preferred). NaOH could be used to adjust the ph of ddh2o. Note: The DNA is ready for downstream applications such as cloning/subcloning, RFLP, library screening, in vitro translation, sequencing, transfection of robust cells such as HEK293 cells. Note: It s highly recommended to remove the endotoxin if the DNA is used for endotoxin-sensitive cell lines, primary cultured cells or microinjection. 12. The DNA concentration can be calculated as, Concentration (µg/ml) = OD260 nm x 50 x dilution factor. 7

9 EZgene TM Plasmid Miniprep II Spin/Vacuum Protocol 1. Set up the vacuum manifold according to manufacture s instruction and connect the column to the manifold. 2. Carry out step 1-6 on Page 6 in previous protocol. 3. Carefully transfer the clear lysate to the DNA column and turn on the vacuum to allow the lysate pass through the column. 4. Add 500 µl Buffer KB into the spin column and allow the lysate pass through the column by vacuum. Note:This step is important to remove residual protein contaminations especially for enda+ strains and be highly recommended for high quality plasmid DNA. 5. Add 650 µl of DNA Wash Buffer to the column and allow the vacuum to draw the liquid through the manifold. Turn off the vacuum. Repeat step Transfer the column, with the lid open, to a 2 ml collection tube and centrifuge for 5-10 minutes.at 13,000 rpm 7. Carefully transfer the spin column into a clean 1.5 ml tube and add µl ddh20 or Elution Buffer into the column and let it stand for 1 minute. Elute the DNA by centrifugation at 13,000 rpm for 1 minute. Reload the eluate into the column and centrifuge again to improve the recovery. Note:It is recommended to use Elution Buffer instead of ddh 20 Note: The DNA is ready for downstream applications such as cloning, RFLP, library screening, in vitro translation, sequencing and transfection of robust cells such as HEK293 cells. 8

10 Purification of Low-Copy-Number Plasmid/Cosmid The yield of low copy number plasmid is normally around µg /ml of overnight culture. For isolating low copy number or medium copy number plasmid DNA, use the following guideline: 1. Culture volume: Use 2 x volumes of the high copy number culture. Use 25 ml for the miniprep II. 2. Use 2 x volumes of the Buffer A1, Buffer B1 and Buffer N1. Additional buffers can be purchased from Abgent. 3. Use same volume of DNA Wash Buffer and Elution Buffer. Purification of plasmid > 12 kb For isolating plasmid DNA > 12 kb, use the following guideline: 1. Culture volume: Use 2 x volumes of the culture. 2. Use 2 x volumes of the Buffer A1, Buffer B1 and Buffer N1. Additional buffers can be purchased from Abgent. 3. Use same volume of DNA Wash Buffer and Elution Buffer. 4. Pre-warm the Elution Buffer at C and let the column stand for 5 mins after adding Elution Buffer. 9

11 I. 实验前准备质粒小提试剂盒 II 简明步骤 ( 详细内容请参考说明书英文部分 ) RNase A: 室温下可稳定贮藏半年, 长期贮藏请置于 4 o C 保存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 4 o C 保存 DNA Wash Buffer*: 使用前请将 60 ml (BG0004) 或 96 ml (BG0005) 或 60 ml (BG0006)96-100% 乙醇加入到每瓶 DNA Wash Buffer 中 Buffer B1: 在低于室温时会沉淀, 请于 50 o C 左右水浴加热至沉淀完全溶解, 溶液澄清, 使用后保证 Buffer B1 瓶盖旋紧在室温下 (22-25 o C) 进行所有离心操作 II. 注意事项质粒拷贝数 : 纯化中低拷贝的质粒时, 使用 2 倍的菌液体积,2 倍的 Buffer A1, B1, N1, 相同体积的 DNA Wash Buffer 和 Elution Buffer. 转化菌 : 若为 -70 o C 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的菌种进行培养 III. 操作步骤若用于提取 1-4 ml 的菌落, 请将 Buffer A1, B1, N1 的量降低至 250 µl, 250 µl 及 350 µl,dna wash Buffer 及 Elution Buffer 的量不变 1. 接种新鲜的单个菌落到 5-12 ml 的 LB 培养基 ( 含适量抗生素 ),37 o C 震荡培养小时 ( 勿超过 16 小时 ) 室温下 10,000 rpm 离心 1 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体裂解不充分, 第 5 步离心后沉淀较松, 不易吸取上清注 : 本说明书中的操作程序适用于标准 LB (Luria Bertani) 培养基培养小时后,OD 600( 细菌密度 ) 在之间的菌液若采用的是富集培养基, 如 TB 或 2 YT, 注意保证 OD600 不超过加入 450 µl Buffer A1( 确保已加入 RNase A), 用移液器或涡流震荡充分悬浮细菌细胞注 : 菌体如果没有充分悬浮均匀, 将导致菌体裂解不完全, 从而使产量降低 3. 加入 450 µl Buffer B1, 轻轻地反转多次至溶液充分混匀, 室温静置 2-10

12 5 分钟至溶液粘稠而澄清注 : 切勿剧烈振荡静置时间不超过 5 分钟, 时间过长会导致基因组 DNA 污染或质粒受到损伤若溶液未清亮澄清, 则表明菌体裂解不充分, 应加大 Buffer B1 的用量或减少菌体量 4. 加入 550 µl Buffer N1, 立即反转多次, 至溶液充分混匀, 此时出现白色絮状沉淀 5. 将离心管转至高速离心机, 在室温下 13,000 rpm 离心 10 分钟 ( 若上清中有白色沉淀, 可再次离心 ) 6. 小心吸取 700 µl 离心后的上清液至带有收集管的 DNA 柱中 ( 避免吸起沉淀 ), 室温下 13,000 rpm 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 6 至全部上清离心过 DNA 柱 7. 向 DNA 柱中加入 500 µl Buffer KB, 室温下 13,000 rpm 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注 : 为了获得高质量的质粒 DNA, 我们推荐使用 Buffer KB, 以确保去除蛋白残留污染 8. 向离心柱中加入 650 µl DNA Wash Buffer( 确保已加入无水乙醇 ), 室温下,13,000 rpm 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 8 9. 将离心柱放回高速离心机中,13,000 rpm 室温下开盖离心 5-10 分钟, 以彻底去除残留的乙醇注 : 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 10. 将离心柱转至一个新的 1.5 ml 离心管中, 向 DNA 柱的正中间加入 100~150 µl( 体积 >100 µl) 的 ddh 2O(pH 在之间 ) 或 Elution Buffer, 室温放置 2 分钟,13,000 rpm 离心 1 分钟, 洗脱质粒 DNA 将洗脱液再加到柱的中间,13,000 rpm 离心 1 分钟, 洗脱质粒 DNA 注 : 我们推荐使用 Elution Buffer 进行洗脱, 质粒 DNA 保存在水中可能不稳定注 : 提取到的质粒 DNA 可直接用于基因克隆测序酶切文库筛选体外转录翻译转染 HEK293 细胞若用于转染内毒素敏感性细胞株, 原代细胞及用于微注射, 建议去除内毒素 11

13 Trouble Shooting Guide Problems Low Yield Possible Reasons Bacterial culture overgrown or not fresh. Suggested Improvements Grow bacterial 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 No DNA Genomic DNA contamination RNA contamination Plasmid DNA floats out of wells while running in agarose gel, DNA doesn t freeze or smell of ethanol Low copynumber plasmid. Plasmid lost in Host E.coli Over-time incubation after adding buffer B1. RNase A not added to solution A1. Trace EtOH not completely removed from column. Increase culture volume and scale up the volume of buffers according to the instruction on page 9. Prepare fresh culture. Do not vortex or mix aggressively after adding Buffer B1. Do not incubate more than 5 minutes after adding Buffer B1. Add RNase A to Buffer A1. Make sure that no ethanol residues remain in the silicon membrane before elute the plasmid DNA. Re-centrifuge or vacuum again if necessary. 12

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摘要

中華民國第五十一屆中小學科學展覽會作品說明書科別 : 生物科組別 : 高中組作品名稱 : 不給糖就搗蛋 - 蛀牙細菌的抑制關鍵詞 :Bacillus horikoshii 轉糖鏈球菌蛀牙編號 : 摘要 : 本實驗的目的是希望能找出人類牙齒上的主要菌