Sep 11, 2020
Mycoplasma
- 1University of California, San Francisco
- Neurodegeneration Method Development CommunityTech. support email: ndcn-help@chanzuckerberg.com
Protocol Citation: Andrea Argouarch 2020. Mycoplasma. protocols.io https://dx.doi.org/10.17504/protocols.io.8gphtvn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 19, 2019
Last Modified: September 11, 2020
Protocol Integer ID: 28911
Keywords: dura mater, dural cells, mycoplasma, bulldog
Abstract
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.
Materials
STEP MATERIALS
Epicentre QuickExtract™ DNA Extraction SolutionEpicentreCatalog #QE09050
Trypsin-EDTA (0.05%), phenol redThermo FisherCatalog #25300062
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
e-Myco PLUS Mycoplasma PCR Detection KitCatalog #25234
AgaroseCatalog #PBSA1705
Ethidium Bromide (1% Solution/Molecular Biology)Fisher ScientificCatalog #BP130210
Gel Loading Dye Blue (6X) - 4.0 mlNew England BiolabsCatalog #B7021S
GeneRuler 1 kb Plus DNA Ladder, ready-to-useThermo FisherCatalog #SM1333
Protocol materials
AgaroseCatalog #PBSA1705
Ethidium Bromide (1% Solution/Molecular Biology)Fisher ScientificCatalog #BP130210
Gel Loading Dye Blue (6X) - 4.0 mlNew England BiolabsCatalog #B7021S
GeneRuler 1 kb Plus DNA Ladder, ready-to-useThermo FisherCatalog #SM1333
Epicentre QuickExtract™ DNA Extraction SolutionEpicentreCatalog #QE09050
Trypsin-EDTA (0.05%), phenol redThermo FisherCatalog #25300062
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
e-Myco PLUS Mycoplasma PCR Detection KitCatalog #25234
Trypsin-EDTA (0.05%), phenol redThermo FisherCatalog #25300062
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
Epicentre QuickExtract™ DNA Extraction SolutionEpicentreCatalog #QE09050
e-Myco PLUS Mycoplasma PCR Detection KitCatalog #25234
AgaroseCatalog #PBSA1705
Ethidium Bromide (1% Solution/Molecular Biology)Fisher ScientificCatalog #BP130210
Gel Loading Dye Blue (6X) - 4.0 mlNew England BiolabsCatalog #B7021S
GeneRuler 1 kb Plus DNA Ladder, ready-to-useThermo FisherCatalog #SM1333
Observations
Observations
Culture should be in at least 3-6 days of subculture after split
During isolation and PCR, use clean gloves, spray down bench, and keep reagents and tubes cold
On ice
Isolation of gDNA
Isolation of gDNA
Isolate cells once confluent, at least 3-6 days after split
After trypsinization for freezing cells, rinse remaining cells in both flasks with 12 mls 12 mL PBS
Trypsin-EDTA (0.05%), phenol redThermo FisherCatalog #25300062
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190250
Spin for 5 mins 00:05:00 at 100 rpm 1000 rpm at 4C 4 °C
Aspirate supernate
Place in ice bucket On ice on bench or store in -80C -80 °C
Add 40 µl 40 µL of Quick Extract to pellet
Epicentre QuickExtract™ DNA Extraction SolutionEpicentreCatalog #QE09050
Pipette up and down and transfer to PCR tubes
a. Label PCR tube with line, date, and P for pellet
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 65 °C for 15 mins 00:15:00
ii. 98C 98 °C for 10 mins 00:15:00
iii. 4C 4 °C to hold
e. Run time around ~30mins 00:30:00
f. Click enter to exit after run
g. Turn off machine and place on ice On ice
Centrifuge PCR tubes for 5 mins 00:05:00 at max 13.4 rpm and transfer SN to new 1.5 mls tubes labeled gDNA, line, and date On ice
a. Tiny pellet ok left in PCR tube
Nanodrop to determine gDNA (pre-PCR) concentration on ice On ice
Store at -20C -20 °C or run PCR
Nanodrop
Nanodrop
Items to bring to nanodrop machine
a. p2 pipette, p2 tips, pen, kimwipes, ice bucket
b. Keep samples on ice On ice
Open the NanoDrop 1000 software and select nucleic acid from the main menu
Clean the nanodrop pedestal with a kimwipe
Add 2 µl 2 µL of nuclease free water to the center of the pedestal and gently close the lever arm
Click ‘OK’ to initialize the spectrophotometer
Select DNA from the dropdown menu
Clean the nanodrop pedestal with a kimwipe, add 2 µl 2 µL of a blank sample, and click ‘Blank’
a. Use quick extract solution as blank
Clean the nanodrop pedestal with a kimwipe, add 2 ul 2 µL of sample and click ‘Measure’
Record the concentration (ng/ul) of the sample on the tube and a table
Record the 260/280 ratio, and the 260/230 ratio
Expected result
260/280 ratio should be above 1.8 (salt contamination)
260/230 ratio should be 1-2
Clean the nanodrop pedestal with a kimwipe in-between samples
Click exit
Leave kimwipe between the pedestal and the lever arm when finished
PCR Reaction
PCR Reaction
Make a 20 µl 20 µL of PCR reaction in provided eMyco Plasma PCR Tube On ice
e-Myco PLUS Mycoplasma PCR Detection KitCatalog #25234
a. QS to 20 µl 20 µL with nuclease free water
b. Add 40 ng 40 ng of sample
i. Can make working stock if concentration is too high
c. Include a negative control with just water (20 µl) 20 µL
d. Include a positive control with provided positive control (1 µl) 1 µL
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 94 °C for 1 min 00:01:00
ii. 35 Cycles
Denature = 94C 94 °C for 30 secs 00:00:30
Anneal = 58C 58 °C 5 for 20 secs 00:00:20
Extend = 72C 72 °C for 1 min 00:01:00
Final Extension = 72C 72 °C for 5 mins 00:05:00
Hold at 4C 4 °C
d. Press enter to run program
g. Run time is around ~1.5hrs 01:30:00
Can prep for agarose gel near the end of the PCR run
Store gDNA samples, at -20C -20 °C
After run, select enter, turn off machine, and place PCR product on ice On ice
Run gel or store at -20C -20 °C
Agarose Gel
Agarose Gel
Wet rubber around gel chamber to help it slide into gel rig
Assemble with rubber on the black edges of the gel rig and place purple comb in the slot on the right
Make 2% Agarose in small beaker with screw cap for a mini rig size.
AgaroseCatalog #PBSA1705
a.1.2 g 1.2 g of agarose with 60 mls 60 mL of 1X TAE (Stock is 20X TAE)
Microwave for 2 mins 00:02:00 with a loose cap and swirl every 30 seconds 00:00:30 until dissolved
When temperature is cool to the touch, add 1:10,000 of Etbr
Ethidium Bromide (1% Solution/Molecular Biology)Fisher ScientificCatalog #BP130210
Safety information
Mutagen - may potentially cause carcinogenic or teratogenic effects
ie add 6 µl 6 µL Etbr per 60 mls 60 mL of agarose
Swirl to mix and add to chamber to set, allow for 10 mins 00:10:00 to cool in cold room 4 °C
Prep samples for gel in PCR tubes on ice On ice
a. 5 µl 5 µL of PCR product
b. 3 µl 3 µL of Nuclease free water
c. 2.6 µl 2.6 µL of 6X loading buffer (2X final concentration from a 6X stock, purple)
Gel Loading Dye Blue (6X) - 4.0 mlNew England BiolabsCatalog #B7021S
i.(6X)(a) = (2X)(8ul); a = 2.6ul
Rotate gel chamber when set, so the lanes are at the top of the chamber
Fill with 1X TAE until the top of the white knob in gel rig and remove comb slowly
Load 4 µl 4 µL of 1kb O’gene plus ladder into first lane
GeneRuler 1 kb Plus DNA Ladder, ready-to-useThermo FisherCatalog #SM1333
Load 11 µl 11 µL of each sample per well
Add lid, black at the top, red at the bottom, both at the right side
Run at 130V for 50 mins 00:50:00
Store PCR product at -20C -20 °C
Dispose Etbr waste in proper container (liquid and solid)