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[Bio101] Preparation of Taq DNA Polymerase
[Bio101] Taq DNA聚合酶的制备   

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Abstract

Materials and Reagents

  1. Dialysis tubing cellulose membrane avg. flat width 33 mm (Sigma-Aldrich, catalog number: D9652 )
  2. E.coli BL21 strain 
  3. Ammonium sulfate (Sigma-Aldrich, catalog number: A4418 )
  4. IPTG
  5. Ampicillin
  6. (NH4)2SO4
  7. 1 M dextrose
  8. 1 M Tris - HCl (pH 7.9)
  9. 0.5 M EDTA (pH 8.0)
  10. KCl
  11. PMSF
  12. Tween 20
  13. NP40
  14. Lysozyme
  15. Glycerol
  16. Triton-X 100
  17. Lysis buffer (see Recipes)
  18. Buffer A (see Recipes)
  19. Pre - lysis buffer (see Recipes)
  20. Storage buffer (see Recipes)
  21. TAQ buffer (see Recipes)
  22. MgCl2 solution (see Recipes)

Equipment

  1. 2 L flask
  2. Centrifuges
  3. Oakridge tubes

Procedure

Day1

  1. Inoculate a single colony of E.coli BL21 strain with Taq plasmid driven by IPTG inducible promoter into 5 ml of LB broth containing 20 μl of ampicillin (100 mg/ml). Incubate at 37 °C overnight with shaking.

Day 2

  1. 7:00 A.M. prepare 500 ml of LB broth containing 40 mg of ampicillin in a 2 L flask, add 500 μl of overnight culture. Incubate at 37 °C for about 11 h with shaking. Grow these cultures to an OD600 of approx. 0.8.
  2. 8:00 P.M., add 62.5 mg of IPTG, incubate at 37 °C for 12 h with shaking.

Day 3

  1. Transfer overnight culture to 500 ml plastic bottle, centrifuge at 4.5 K for 10 min at 4 °C. Decant supernatant, and resuspend the prep in 50 ml of Buffer A.
  2. Centrifuge at 4.5 K for 10 min at 4 °C, decant supernate, and resuspend the prep in 25 ml of pre-lysis buffer.
  3. Incubate at room temperature (RT) for 15 min.
  4. Add 25 ml of lysis buffer, mix well, transfer prep to Pyrex flask, and incubate at 75 °C for 1 h.
  5. Divide the prep into two Oakridge tubes, cetrifuge at 15,000 rpm for 10 min at 4 °C.
  6. Transfer supernate to clean flask, add 15 g of (NH4)2SO4, stir rapidly at RT for 1/2 h.
  7. Centrifuge at 15,000 rpm for 10 min at 4 °C.
  8. Decant supernate, resuspend prep in 10 ml of Buffer A.

Day 3 & 4

  1. The resuspended protein was dialyzed against 3 changes of storage buffer at 4 °C. (One 7 h, one 12 h and one 7 h, each with> 800 ml of storage buffer. The volume of the protein was reduced to about 5 ml after the dialysis.)
  2. Protein was diluted 1:1 with sterilized storage buffer, and store at -80 °C.

Recipes

  1. Buffer A
    50 mM Tris - HCl (pH 7.9)         25 ml of 1 M Tris - HCl (pH 7.9)
    50 mM dextrose                        25 ml of 1 M dextrose
    1 mM EDTA (pH 8.0)                1 ml of 0.5 M EDTA (pH 8.0)
    q.s. to 500 ml with ddH2O, autoclave, store at RT.
  2. Lysis buffer
    10 mM Tris - HCl (pH 7.9)        1 ml of 1 M Tris-HCl (pH 7.9)
    50 mM KCl                               5 ml of 1 M KCl
    1 mM EDTA (pH 8.0)                0.2 ml of 0.5 M EDTA (pH 8.0)
    1 mM PMSF*                            2 ml of 50 mM PMSF
    0.5% Tween 20                       0.5 ml Tween 20
    0.5% NP40                             0.5 ml NP40
    q.s. to 100 ml with ddH2O, autoclave, store at RT.
    * Toxic material, always wear a mask and gloves when dealing with it!
    PMSF can only be dissolved in 100% EtOH.
  3. Pre - lysis buffer
    Buffer A plus 4 mg/ml lysozyme.
  4. Storage buffer
    50 mM Tris - HCI (pH 7.9)         150 ml of 1 M Tris- HCI (pH 7.9)
    50 mM KCl                                150 ml of 1 M KCl
    0.1 mM EDTA (pH 8.0)              0.6 ml of 0.5 M EDTA (pH 8.0)
    1 mM DTT                                 3 ml of 1 M DTT
    0.5 mM PMSF*                          30 ml of 0.5 mM PMSF
    50% glycerol                            1.5 L of glycerol
    q.s. to 3 L w/ D.D. H2O, autoclave, store at RT.
  5. TAQ buffer
    500 mM KCl                        5 ml of 1 M KCl
    100 mM Tris                        1 ml of of 1 M Tris (pH 9.0)
    1% Triton                           100 μl of Triton-X 100
    Bring to a final volume of 10 ml. Autoclave and aliquot into 1.5 ml tubes. Store at -20 °C.
  6. MgCl2 solution
    Add 0.0238 g of MgCl2 to 10 ml of ddH2O. Autoclave and divide into 1.5 ml tubes, store at -20 °C.

References

  1. Pluthero, F. G. (1993). Rapid purification of high-activity Taq DNA polymerase. Nucleic Acids Res 21(20): 4850-4851.

简介


材料和试剂

  1. 透析管纤维素膜平均 平宽33mm(Sigma-Aldrich,目录号:D9652)
  2. 大肠杆菌 BL21 strain 
  3. 硫酸铵(Sigma-Aldrich,目录号:A4418)
  4. IPTG
  5. 氨苄青霉素
  6. (NH 4)2 SO 4 SO 4
  7. 1M葡萄糖
  8. 1M Tris-HCl(pH7.9)
  9. 0.5M EDTA(pH8.0)
  10. KCl
  11. PMSF
  12. 吐温20
  13. NP40
  14. 溶菌酶
  15. 甘油
  16. Triton-X 100
  17. 裂解缓冲液(见配方)
  18. 缓冲液A(参见配方)
  19. 预裂解缓冲液(见配方)
  20. 存储缓冲区(参见配方)
  21. TAQ缓冲区(参见配方)
  22. MgCl 2溶液(参见配方)

设备

  1. 2 L烧瓶
  2. 离心机
  3. Oakridge管

程序

Day1

  1. 用由IPTG诱导型启动子驱动的Taq质粒将大肠杆菌BL21菌株的单个菌落接种到含有20μl氨苄青霉素(100mg/ml)的LB肉汤中。 在37℃下振荡孵育过夜。

第2天

  1. 7:00 AM。 在2L烧瓶中制备500ml含40mg氨苄青霉素的LB肉汤,加入500μl过夜培养物。 在37℃下振荡孵育约11小时。 将这些培养物培养至约600℃的OD 600。 0.8。
  2. 8:00 P.M.,加入62.5mg IPTG,在37℃下振荡孵育12小时。

第3天

  1. 转移过夜培养到500毫升塑料瓶,在4.5 K在4℃离心10分钟。 倾析上清液,并将制备物重悬在50ml缓冲液A中。
  2. 在4℃下在4.5K离心10分钟,倾析上清液,并将制备物重新悬浮在25ml预裂解缓冲液中。
  3. 在室温(RT)孵育15分钟。
  4. 加入25ml裂解缓冲液,混匀,转移制备到Pyrex烧瓶,并在75°C孵育1小时。
  5. 将制备物分成两个Oakridge管,在4℃下以15,000rpm离心10分钟。
  6. 将转移上清液至干净的烧瓶中,加入15g(NH 4)2 SO 4 SO 4,在室温下快速搅拌1/2小时。
  7. 在4℃下以15,000rpm离心10分钟。
  8. 滗析上清液,重悬于10ml缓冲液A中制备

第3天& 4

  1. 将重悬的蛋白质在4℃下对储存缓冲液的3次变化进行透析。 (一次7小时,一次12小时和一次7小时,每次具有> 800ml储存缓冲液,透析后蛋白质的体积减少至约5ml)
  2. 蛋白质用无菌储存缓冲液1:1稀释,并储存在-80℃

食谱

  1. 缓冲区A
    50 mM Tris-HCl(pH 7.9)        25ml 1M Tris-HCl(pH7.9) 50 mM葡萄糖                       25ml的1M葡萄糖 1 mM EDTA(pH 8.0)                 1ml 0.5M EDTA(pH8.0)
    适量至500ml,使用ddH 2 O,高压釜,在室温储存
  2. 裂解缓冲液
    10 mM Tris-HCl(pH 7.9)        1ml的1M Tris-HCl(pH7.9)
    50毫克KCl                                5ml的1M KCl
    1 mM EDTA(pH 8.0)                 0.2ml 0.5M EDTA(pH8.0) 1 mM PMSF *                            2ml 50mM PMSF 0.5%吐温20                      0.5ml吐温20 0.5%NP40                             0.5 ml NP40
    适量至100ml,用ddH 2 O,高压釜,在室温下储存 *有毒物质,在处理时总是戴上口罩和手套!
    PMSF只能溶于100%EtOH中
  3. 预裂解缓冲液
    缓冲液A加4mg/ml溶菌酶
  4. 存储缓冲区
    50 mM Tris-HCl(pH 7.9)         150ml 1M Tris-HCl(pH 7.9)
    50毫克KCl                                 150ml的1M KCl
    0.1 mM EDTA(pH 8.0)               0.6ml 0.5M EDTA(pH8.0)
    1 mM DTT                                 3ml 1M DTT
    0.5 mM PMSF *                          30ml 0.5mM PMSF 50%甘油                            1.5升甘油 适量至3L w/D.D。 H 2 O,高压釜,在室温储存。
  5. TAQ缓冲区
    500毫克KCl                         5ml的1M KCl
    100 mM Tris                        1ml的1M Tris(pH9.0)
    1%Triton                       100μlTriton-X 100
    最终体积为10ml。高压灭菌并等分到1.5ml管中。储存于-20°C。
  6. MgCl 2溶液
    将0.0238g MgCl 2加入10ml ddH 2 O中。高压灭菌并分成1.5ml管,-20℃保存

参考文献

  1. Pluthero,F.G。(1993)。 快速纯化高活性的 DNA聚合酶。 Nucleic Acids Res 21(20):4850-4851。
  • English
  • 中文翻译
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zheng, W. (2011). Preparation of Taq DNA Polymerase. Bio-protocol Bio101: e136. DOI: 10.21769/BioProtoc.136;
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quan jiang
the university of hong kong
"home-made Taq DNA polymerase"
It is delighted reading your protocol regarding home-made Taq Polymerase purification. . I must say that it is good news for a small lab with limited budget. I would like to make my own lab DNA polymerase. Would you send clone to me at your convenience?
5/16/2012 9:39:41 PM Reply
Fanglian He
University of Pennsylvania

You can request Taq polymerase plasmid from Addgene, http://www.addgene.org/25712/.

5/20/2012 2:49:56 PM