TITLE: Mycoplasma
AUTHORS: Andrea Argouarch

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Detection of mycoplasma utilizing the bulldog-bio kit. Steps include, gDNA isolation from cells, nanodrop, PCR, and running an agarose gel to detect mycoplasma within a cell culture line. </div><div class = "text-block"><span>Image from </span><a href="https://cellculturedish.com/cell-culture-basics-mycoplasma-101-a-practical-guide-to-prevention-detection-and-elimination-of-mycoplasma-contamination" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://cellculturedish.com/cell-culture-basics-mycoplasma-101-a-practical-guide-to-prevention-detection-and-elimination-of-mycoplasma-contamination/</span></a></div></div>

[STEPS]
?. [Nanodrop]
Items to bring to nanodrop machine          a. p2 pipette, p2 tips, pen, kimwipes, ice bucket       b. Keep samples on ice
on ice
?. [Nanodrop]
Open the NanoDrop 1000 software and select nucleic acid from the main menu
?. [Nanodrop]
Clean the nanodrop pedestal with a kimwipe
?. [Nanodrop]
Add 2 µl   of nuclease free water to the center of the pedestal and gently close the lever arm
2 µl
?. [Nanodrop]
Click ‘OK’ to initialize the spectrophotometer
?. [Nanodrop]
Select DNA from the dropdown menu
?. [Nanodrop]
Clean the nanodrop pedestal with a kimwipe, add 2 µl   of a blank sample, and click ‘Blank’      a. Use quick extract solution as blank
2 µl
?. [Nanodrop]
Clean the nanodrop pedestal with a kimwipe, add 2 ul   of sample and click ‘Measure’
2 µl
?. [Nanodrop]
Record the concentration (ng/ul) of the sample on the tube and a table
?. [Nanodrop]
Record the 260/280 ratio, and the 260/230 ratio
260/280 ratio should be above 1.8 (salt contamination)260/230 ratio should be 1-2
?. [Nanodrop]
Clean the nanodrop pedestal with a kimwipe in-between samples
?. [Nanodrop]
Click exit
?. [Nanodrop]
Leave kimwipe between the pedestal and the lever arm when finished
?. [Observations]
Culture should be in at least 3-6 days of subculture after split
?. [Observations]
During isolation and PCR, use clean gloves, spray down bench, and keep reagents and tubes cold
on ice
?. [Isolation of gDNA]
Isolate cells once confluent, at least 3-6 days after split
?. [Isolation of gDNA]
After trypsinization for freezing cells, rinse remaining cells in both flasks with 12 mls   PBS
12 mL
?. [Isolation of gDNA]
Spin for 5 mins   at 100 rpm   at 4C
Centrifuge: 1000 33
4 °C
?. [Isolation of gDNA]
Place in ice bucket  on bench or store in -80C
on ice
-80 °C
?. [Isolation of gDNA]
Add 40 µl   of Quick Extract to pellet
40 µl
?. [Isolation of gDNA]
Pipette up and down and transfer to PCR tubes     a. Label PCR tube with line, date, and P for pellet
?. [Isolation of gDNA]
Extract gDNA with thermocycler program    a. Turn on Power button on the back of machine    b. Open lid with handle     c. Put PCR tubes in small slot and close lid    d. Select File, click Enter, select pcr chamber and start program         i. 65C   for 15 mins         ii. 98C   for 10 mins         iii. 4C   to hold     e. Run time around ~30mins     f. Click enter to exit after run    g. Turn off machine and place on ice
65 °C
98 °C
4 °C
on ice
?. [Isolation of gDNA]
Centrifuge PCR tubes for 5 mins   at max   and transfer SN to new 1.5 mls tubes labeled gDNA, line, and date     a. Tiny pellet ok left in PCR tube
Centrifuge: 13.4 33
on ice
?. [Isolation of gDNA]
Nanodrop to determine gDNA (pre-PCR) concentration on ice
on ice
?. [Isolation of gDNA]
Store at -20C   or run PCR
-20 °C
?. [PCR Reaction]
Make a 20 µl   of PCR reaction in provided eMyco Plasma PCR Tube      a. QS to 20 µl   with nuclease free water     b. Add 40 ng   of sample         i. Can make working stock if concentration is too high     c. Include a negative control with just water (20 µl)     d. Include a positive control with provided positive control (1 µl)
20 µl
on ice
20 µl
40 ng
20 µl
1 µl
?. [PCR Reaction]
Run PCR program     a. Turn on machine at the back     b. Tap to mix, place samples into thermocycler in small tube holes, and close lid    c. Run myco program          Initial Denature = 94C   for 1 min                 ii. 35 Cycles        Denature = 94C   for 30 secs         Anneal = 58C  5 for 20 secs         Extend = 72C   for 1 min         Final Extension = 72C   for 5 mins         Hold at 4C     d. Press enter to run program    g. Run time is around ~1.5hrs
94 °C
94 °C
58 °C
72 °C
72 °C
4 °C
?. [PCR Reaction]
Can prep for agarose gel near the end of the PCR run
?. [PCR Reaction]
Store gDNA samples, at -20C
-20 °C
?. [PCR Reaction]
After run, select enter, turn off machine, and place PCR product on ice
on ice
?. [PCR Reaction]
Run gel or store at -20C
-20 °C
?. [Agarose Gel]
Wet rubber around gel chamber to help it slide into gel rig
?. [Agarose Gel]
Assemble with rubber on the black edges of the gel rig and place purple comb in the slot on the right
?. [Agarose Gel]
Make 2% Agarose in small beaker with screw cap for a mini rig size.    a.1.2 g   of agarose with 60 mls   of 1X TAE (Stock is 20X TAE)
1.2 g
60 mL
?. [Agarose Gel]
Microwave for 2 mins   with a loose cap and swirl every 30 seconds   until dissolved
?. [Agarose Gel]
When temperature is cool to the touch, add 1:10,000 of Etbr     ie add 6 µl  Etbr per 60 mls   of agarose
Mutagen - may potentially cause carcinogenic or teratogenic effects
6 µl
60 mL
?. [Agarose Gel]
Swirl to mix and add to chamber to set, allow for 10 mins    to cool in cold room
4 °C
?. [Agarose Gel]
Prep samples for gel in PCR tubes on ice     a. 5 µl   of PCR product     b. 3 µl   of Nuclease free water     c. 2.6 µl   of 6X loading buffer (2X final concentration from a 6X stock, purple)        i.(6X)(a) = (2X)(8ul); a = 2.6ul
on ice
5 µl
3 µl
2.6 µl
?. [Agarose Gel]
Rotate gel chamber when set, so the lanes are at the top of the chamber
?. [Agarose Gel]
Fill with 1X TAE until the top of the white knob in gel rig and remove comb slowly
?. [Agarose Gel]
Load 4 µl  of 1kb O’gene plus ladder into first lane
4 µl
?. [Agarose Gel]
Load 11 µl  of each sample per well
11 µl
?. [Agarose Gel]
Add lid, black at the top, red at the bottom, both at the right side
?. [Agarose Gel]
Run at 130V for 50 mins
?. [Agarose Gel]
Store PCR product at -20C
-20 °C
?. [Agarose Gel]
Dispose Etbr waste in proper container (liquid and solid)
?. [Isolation of gDNA]
Aspirate supernate