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PROTOCOLS

Table of contents

  1. Molecular biology
    1.1 DNA

    1.2 RNA

    1.3 Proteins

    1.4 Transformations

  2. Stocks and sample preparations
    2.1 Freezer stocks

  3. Assays

    3.1 Competition assays

    3.2. Predation assays

  4. Other protocols

    4.1 Aggregates isolation

    4.2 Myxobacteria Isolation from Soil

1. Molecular biology

DNA

Genomic DNA from QIAGEN kit

For detailed protocol, refer to the handbook QIAGEN Genomic DNA Handbook

NB: This protocol works well with cells from liquid cultures. However, it does not work with spores nor with cells at high densities harvested directly from plates.

  1. Add 2 µl of RNase A (100 mg/ml) to 1 ml of Buffer B1.
  2. Pellet cells at 12000 rpm for 5 min and discard supernatant.
  3. Resupsend bacteria pellet in 1 ml of Buffer B1 with RNase. Gently pippette up and down and then invert tube without vortexing.
  4. Add 80 µl of Lysozyme (100 mg/ml) and 45 µl of Prot K (20 mg/ml). Incubate for at least 45 min at 37 °C while roughly every 10 min gently invert the tube - DO NOT VORTEX.
  5. Transfer solution to a 15 ml falcon tube and add 1.2 ml of Buffer B2 and mix by inverting the tube multiple times. Incubate at 50 °C for 30 min.
  6. Place Genomic-tip 20/G column on vaccum pump holder (previously washed and dried). Equilibrate column with 1 ml of QBT Buffer and allow column to empty by gravity flow.
  7. Invert tube containing sample multiple times and apply to column. Allow to enter the resin by gravity flow or apply vaccum (do not exceed 10 kPa).
  8. Wash column 3x with 1 ml of QC Buffer. Try to maintain column resin humid and do not leave it to dry.
  9. Elute DNA with 2 ml of QF Buffer preheated at 50 °C. Typically this step run O/N gives the best yields.
  10. Precipitate DNA by adding 1.2 ml of isopropanol. Gently invert tube 20 times.
  11. Spool DNA with glass probes if the starting material was sufficiently aboundant (DNA is visibile with neked eye). Otherwise continue form step 13.
  12. Transfer pooled DNA in TE or EB buffer depending on following application.
  13. Centrifuge at 4°C at 9000 rcf for 10 min.
  14. Remove supernatant, add cold Et-OH 70 % and centrifuge again at 9000 rcf for 10 min. Gently discard supernatant and let air dry for about 15 min. Resuspend pellet in TE or EB buffer (typically 50 µl) depending on the following applicaiton.

Swarms DNA extraction (suitable for both Long and Short-Read seq)

This protocol is based on the PowerBiofilm QIAGEN kit.
NB for long-read seq: Instead of gently invert tubes to preserve long DNA fragments.

  1. Spin down cells at 14000 rpm.
  2. Remove supernatant and add 350 µl of MBL media. Transfer to the PowerBiofilm Tube.
  3. Add 100 µl of FB solution and vortex briefly.
  4. Incubate PowerBiofilm Bead Tube at 65 °C for 5 min.
  5. Use Bead Ruptor at GDC:
    • 1 Cycle
    • 30 sec
    • 5.30 power (~ 3200 rpm)
  6. Centrifuge at 14000 rpm for 1 min. Transfer supernatant in a 2 ml tube.
  7. Add 100 µl of IRS solution and store at 4 °C for 5 min.
  8. Centrifuge at 14000 rpm for 1 min at RT.
  9. Avoiding the pellet, transfer all supernatant to a 2 ml. Then add 900 µl of MR solution and vortex briefly.
  10. Load 650 µl of supernatant to a MB Spin-Column and centrifuge at 14000 rpm for 1 min at RT. Discard flow-through and repeat until the all supernatant had been processed.
  11. Place MB Spin-Col into a new 2 ml collection tube.
  12. Add 650 µl of PW solution and centrifuge at 14000 rpm for 1 min.
  13. Discard flow-through and add 650 µl of Et-OH and centrifuge at 14000 rpm for 1 min.
  14. Discard flow-through and centrifuge at 14000 rpm for 2 min again.
  15. Place the MB Spin-Column in a new 2 ml collection tube.
  16. Add 100 µl of EB solution to the center of the column.
  17. Cetrifuge at 14000 rpm for 1 min.
  18. Discard column and store the DNA at 4 °C or -20 °C according to application.

Chelex

  1. Spin down cells at 12000 rpm for 5 min.
  2. Remove supernatant and add 250 µl of Chelex-TE solution pH 8.0. Resuspend pellet by pippetting not too vigorously to avoid DNA shearing.
  3. Incubate samples for 25 min at 80 °C.
  4. Heat at 90 °C for 10 min.
  5. Incubate in ice for 5 min.
  6. Spin down at 13000 rpm for 3 min.
  7. Remove supernatant.
  8. Store at 4 °C or -20 °C according to following procedure.

Spore DNA extraction (suitable for both Short-Read seq)

FISH

Colony PCR

RNA

Total RNA extraction

Northern blot

Protein

Quick extraction

Acidic extraction

Western blot

Transformations

Targeted insertion

Allele swap

Stocks and sample preparations

Freezer stocks

From Liquid Cultures