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Mycoplasma test

Recipe Created on 10 Feb 2015

Authors

Agnieszka Pastula

Procedure

1. Collect 0.5 ml supernatant from cells and put it into Eppendorf tube. Label the tube.

2. Centrifuge the supernatant in an Eppendorf tube at 200g for 1 min at room temperature to pellet cell debris.

3. Prepare Master Mix

 Component

 Volume (ul) per one reaction

 Red Taq PCR Master Mix 2x (Sigma)

6.25

 H2O

4.75

 Primer Mix 10 uM (r + f)

0.5

 Supernatant

0.5

 Total

12.5

 

 

 

 

 

 

 

Include:

  • positive control
  • negative control (NC) -> water instead of supernatant.

Use filtered tips!

4. PCR conditions

1.

95°C

5 min

2.

94°C

30 sec

3.

55°C

30 sec

4.

72°C

1 min

5.

72°C

10 min

6.

4°C

 

 

 

 

 

 

 

 

 Steps 2-4 -> 40 cycles

5. Electrophoresis -> 2% agarose gel; expected product size: 270 bp

Primers

Mycopl f  GGGAGCAAACAGGATTAGATACCCT

Mycopl r  TGCACCATCTGTCACTCTGTTAACCTC

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