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DNA was extracted from the supernatant of each sample. After PCR-amplification of mycoplasma DNA, detection was performed by gel electrophresis. The PCR primers were designed to cover the consensus sequences that can detect all types of mycoplasma species.

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[Bio101] Cell Culture Mycoplasma Detection by PCR
[Bio101] 用PCR检测细胞培养支原体属

分子生物学 > DNA > PCR
作者: Huan Pang
Huan PangAffiliation: Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, USA
For correspondence: pang_huan@hotmail.com
Bio-protocol author page: a48
4/20/2012, 7157 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.207

[Abstract] DNA was extracted from the supernatant of each sample. After PCR-amplification of mycoplasma DNA, detection was performed by gel electrophresis. The PCR primers were designed to cover the consensus sequences that can detect all types of mycoplasma species.

[Abstract] DNA通过每个样品的上清提取。PCR扩增支原体属的DNA后,用电泳检测。PCR引物设计应是支原体属每一个种都有的保守序列。

Materials and Reagents

  1. Ampli Taq Gold (Life Technologies, InvitrogenTM, catalog number: 4338856 )
  2. Sodium acetate
  3. Ethanol
  4. Phenol
  5. MgCl2
  6. PCR buffer
  7. Sodium acetate
  8. dNTPs
  9. Agarose gel
  10. TE-saturated phenol
  11. Ethidium bromide
  12. DDW
  13. Stock solution A (see Recipes)
  14. Master mixture A (see Recipes)

Equipment

  1. 1.5 ml Eppendorf tube
  2. Centrifuges
  3. Micropipette  

Procedure

  1. Preparation of template DNA
    1. From cultured supernatant (in the case of cells)
      1. The sample aliquots (600 μl/sample) are obtained from the supernatant of the sample cells in the chamber. We use one 1.5 ml Eppendorf tube per sample.
      2. Add same amount of TE-saturated phenol (600 μl) to each eppendorf tube and mix vigously by vortex for several seconds.
      3. Centrifuge at 15,000 rpm for 5 min at room temperature (RT).
      4. Transfer the 400 μl supernatant to a new eppendorf tube and add 10 μl of 3 M sodium acetate.
      5. Mix well and spin down the aliquots.
      6. Add 2.5 times volume of absolute ethanol (1 ml), mix well, and keep at -80 °C for 15 min.
      7. Centrifuge at 15,000 rpm for 10 min at 4 °C.
      8. Discard the supernatant by micropipette and rinse with 80% ethanol.
      9. Centrifuge at 15,000 rpm for 10 min at 4 °C.
      10. Discard the supernatant by micropipette completely and air-dry.
      11. Dissolve in 40 μl DDW by vigorous vortexing and use this as the template DNA for PCR.

    2. From frozen ampule of sample cells
      1. Thaw the frozen ampule at RT.
      2. Open the ampule and take 600 μl of cell suspension to a 1.5 ml Eppendorf tube.
      3. Add same amount of TE-saturated phenol (600 μl) to each eppendorf tube and mix vigorously by vortex for several seconds. From this step, perform all the same procedures described in A. 2-11.
        Note: In this protocol the final 40 μl dissolved solution becomes quite viscous because of containing large amounts of genomic DNA from the sample cells. Therefore, vortex mixing is needed for a longer time.

  2. PCR
    Reaction mixture per sample (per one tube)
    50.0 μl
    DDW
    29.75 μl
    Stock solution A
    15.0 μl
    Template DNA
    5.0 μl
    Ampli Taq Gold(5 U/μl)
    0.25 μl
    Total
    1. Beforehand we make "Stock solution A” and "Master mixture A” for 10 tubes as recipes. Distribute 45 μl of "Master mixture A" to each tube.
    2. Add 5.0 μl of template DNA, mix well, and spin down.
    3. Perform PCR cycling by the thermal cycler machine as following schedule:
      95 °C
      9 min

      94 °C
      30 sec*

      55 °C
      2 min *
      *30 cycles repeated
      72 °C
      2 min *

      72 °C
      5 min

    4. Store the samples at 4 °C.

  3. Agarose gel electrophoresis
    1. 10 μl of PCR products are loaded onto 2% agarose gel.
    2. Stain the gel with ethidium bromide (0.1 μg/ml) for 10 min and take photograph under UV light.
      Note: If you know that the sample was highly contaminated by mycoplasma, you can use the supernatant from cultured sample cells directly for the PCR reaction mixture. In this case 3-5 μl of the supernatant will be applicable to the reaction mixture of PCR without all the above sample preparation procedures.

Recipes

  1. Stock solution A (15 μl for 1 sample)
    10x PCR buffer
    5 μl
    MgCl2 (25 mM)
    4 μl
    dNTPs (each 2.5 mM)
    4 μl
    Primer F1 (10 pmol/μl)
    1 μl
    Primer R1 (10 pmol/μl)
    1 μl
    Total
    15 μl
  2. Master mixture A (45 μl per tube, for 10 samples)
    DDW
    312.4 μl
    Stock solution A
    157.5 μl
    Ampli. Taq Gold
    2.6 μl
    Total
    472.5 μl

References

  1. Harasawa, R., Mizusawa, H., Nozawa, K., Nakagawa, T., Asada, K. and Kato, I. (1993). Detection and tentative identification of dominant mycoplasma species in cell cultures by restriction analysis of the 16S-23S rRNA intergenic spacer regions. Res Microbiol 144(6): 489-493.

材料和试剂

 

1.         Sodium acetate

2.         Ethanol

3.         Phenol

4.         Ampli Taq Gold (Applied Biosystems, catalog number: 4338856)

5.         MgCl2

6.         PCR Buffer

7.         dNTPs

8.         Agarose gel

9.         Ethidium bromide

 

设备

 

1.         1.5ml eppendorf tube

2.         离心机

 

步骤

 

1.         准备DNA模板

1)       从培养的上清(in case out of cells

a.       样品等份(600μl/sample)是在从chamber中每个样品的上清得到。事实上,我们每个样品用1.5ml管。

b.      加入等体积TE-饱和酚到每个管中。并通过涡旋大力混合数秒钟。

c.       室温下15000rpm离心 5 min

d.      转移上清400μl到一个新的管中,加入10μl 3M乙酸钠

e.       混匀,离下管中等份部分。

f.        加入2.5倍体积的无水乙醇(1ml),混匀,-80°C15分钟。

g.      4°C 15000rpm 离心10分钟。

h.       用枪吸走上清,用80%漂洗。

i.         4°C 15000rpm 离心10分钟。

j.         用枪完全吸走上清,空气晾干。

k.       40μl双蒸水大力涡旋溶解,用它作为PCR模板DNA

2)       从样品细胞的冰冻ampule

a.       在室温下融合冰冻的ampule

b.      打开ampule吸取600μl细胞悬浮液到管中。

c.       加同体积TE-饱和酚(600μl)到管中,通过涡旋大力混匀数秒钟。从这步起,按照A. 2-11进行所有的步骤。

 

提示:这个方法最终的40μl溶解的溶液会有一些粘稠,因为样品细胞中含有大量的基因组DNA。以此,涡旋混合需要更长的时间。

2.         PCR

每个样品反应混合物(每管)

DDW                                        29.75μl

Stock solution A                       15.0 μl

Template DNA                           5.0 μl

Ampli Taq Gold(5U/μl)               0.25 μl

Total                                         50.0 μl

1)       事先,我们制"Stock solution A”"Master mixture A” 10管作为试剂。分配45μl "Master mixture A"到每个管中。

2)       加入5ulDNA模板,混匀,离心。

3)       通过热循环仪如下设计PCR程序。

95°C    9 min

94°C    30 sec*

55°C    2 min *    *30 cycles repeated

72°C    2 min *      

72°C    5 min

4)       Store the samples at 4°C. 将样品放到4°C

3.         琼脂糖凝胶电泳

1)       10 μlPCR产物加到2%琼脂糖胶中。

2)       EB (0.1μg/ml)染胶十分钟,在UV灯下照相。

 

注意:如果你知道样品被支原体属高度污染,你可以将培养的样品细胞的上清直接加到PCR反应混合物中。如果这样的话,3-5μl上清就可以用于PCR反应的混合物了,无需上述样品制备步骤。

 

试剂

 

1.         Stock solution A (15 μl for 1 sample)

10xPCR Buffer                          5 μl

MgCl2 (25mM)                           4 μl

dNTPs (each 2.5mM)                 4 μl

Primer F1 (10pmol/μl)                1 μl

Primer R1 (10pmol/μl)                1 μl

Total                                         15 μl

2.         Master mixture A (45μl per tube, for 10 samples)

DDW                                        312.4 μl

Stock solution A                       157.5 μl

Ampli. Taq Gold                        2.6 μl

Total                                         472.5 μl

 

References

 

1.         Harasawa R., Mizusawa H., Nozawa K., Nakagawa T., Asada K., Kato I. (1993). Detection and tentative identification of dominant mycoplasma species in cell cultures by restriction analysis of the 16S-23S rRNA intergenic spacer regions. Research in Microbiology 144(6): 489-93. 

 

 

 

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How to cite this protocol: Pang, H. (2012). Cell Culture Mycoplasma Detection by PCR. Bio-protocol Bio101: e207. DOI: 10.21769/BioProtoc.207; Full Text



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