TITLE: CTAB extraction of DNA and RNA of respiratory samples for microbial work
AUTHORS: Leah Cuthbertson, Michael Cox, Phillip  James , Colin Churchward, Bill Cookson, Miriam Moffatt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">This extraction method is modified from Griffiths et al 2000 Applied Environmental Microbiology 66(12): 5488–5491  and DeAngelis et al 2009 ISME Journal 3 pp 168-178.</span><span></span></div><div class = "text-block"><span style = "font-style:italic;">In comparison with DNA extraction kits, nucleic acid yields tend to be higher, though it is more laborious for larger numbers of samples.The extraction uses CTAB and Phenol:Chloroform:Isoamyl alcohol for lysis and the precipitation of nucleic acid is PEG based. An alternative to bead-beating is described, which should only be used for extracting DNA from cultured isolates. Bead-beating is avoided in this case in order to minimise shearing of DNA prior to genomic DNA sequencing.For all other samples use bead-beating is recommended.</span></div><div class = "text-block"><span style = "font-weight:bold;:UNDERLINE;">This Protocol will cover the extraction of the following sample types;</span></div><div class = "text-block">Bacterial isolates</div><div class = "text-block">Upper respiratory tract swabs (nasal and throat swabs)</div><div class = "text-block">Upper respiratory tract lavage and aspirates (nasopharyngeal aspirate, endotracheal aspirate, nasal lavage, Mouth wash)</div><div class = "text-block">Saliva</div><div class = "text-block">Sputum (expectorated spontaneously produced, and induced sputum)</div><div class = "text-block">Upper and lower respiratory tract synthetic absorptive matrix (SAM) strips</div><div class = "text-block">Lower respiratory tract samples obtained via bronchoscopy - lung brushes, bronchoalveolar lavage and pleural fluid</div><div class = "text-block">Lung tissue – human or mouse</div><div class = "text-block">Lung biopsies</div><div class = "text-block">Cerebral spinal fluid (CSF)</div><div class = "text-block">This is a double extraction protocol, you are adding a second volume of Phenol:Chloroform:Isoamyl alcohol to each sample, precipitating two aqueous phases per sample and then recombining the resulting DNA pellets.This approach significantly increases the yield of DNA from samples and strains.</div></div>

[STEPS]
?. [Extraction]
Add   aluminium ammonium sulfate to each Lysing Matrix E (LME) tube
50 µl
It is helpful to write the sample numbers on the lid and side of the LME tubes as the beadbeater can rub the numbers off.
?. [Extraction]
Aseptically transfer sample to the LME tube and add 500ul of CTAB to the LME tube and incubate for .
Bacterial Isolates    a. Grow up bacterial isolates overnight in appropriate culture broth    b. Spin down   of broth for   at 16,000 x g    c. In a safety cabinet add 500ul of CTAB and resuspend the pellet. Incubate at room temperature            for  .    d. Bacteria and CTAB can be stored frozen prior to extraction or extracted after   incubation.No additional CTAB is required.Upper respiratory tract swabs (nasal and throat swabs)    a. Prepare 1 sterile spin baskets in a   trefflab tube per sample.    b. 1 pair of autoclaved scissors will be required per swab to transfer swab tips into bead beating     tubes    c. Keep swabs on ice until transferred into LME tubes    d. Aseptically transfer swab tips into the LME tubes using sterile scissors.    e. Add   of CTAB to the LME tube and incubate for  . Upper respiratory tract lavage and aspirates (nasopharyngeal aspirate, endotracheal aspirate, nasal lavage)    a. Spin a minimum volume of  , optimal volume 5mls of lavage fluid for   at full speed    b. Carefully resuspend pellet in   CTAB extraction buffer and transfer to LME tube.NOTE:For saliva or mouth wash samples and sputum samples pre-alequoting using wide bore tips prior to storage is recommended.Saliva or Mouth wash samples    a. Transfer a known volume of no more than  into a LME tube.Sputum (expectorated spontaneously produced, and induced sputum)    a. Transfer a known volume of no more than   or individual sputum plug, into a LME tube.Upper and lower respiratory tract synthetic absorptive matrix (SAM) strips    a. 1 pair of autoclaved scissors will be required per SAM to transfer SAM into LME tubes    b. Keep SAMs on ice until transferred into LME tubesLung brushes    a. Keep lung brushes on ice until transferred to LME tubesBronchoalveolar lavage or Pleural fluid    a. Spin a minimum volume of  , optimal volume   of lavage fluid for   at full speed    b. Carefully resuspend pellet in  CTAB extraction buffer before transfering to LME tube.NO further CTAB is requiredLung tissue- Human or Mouse    a. Keep tissue on ice until transferred to LME tubesLung biopsies    a. Keep tissue on ice until transferred to LME tubes.CSF    a.Maximise and standardise sample volume across the study.    b.Spin fluid for   at 16,000 x g    c.Carefully suspend pellet in  CTAB extraction buffer before transferring to LME tube.NO further CTAB is required
Transfer of patient samples should be performed in a class 2 safety hood.
?. [Extraction]
Moving to a fume cupboard, carefully and immediately add  of P:C:I (25:24:1).
500 µl
Pre-wet the pipette to avoid drips due to vapour pressure in tip.
Perform this step in a fume hood
Wear double gloves when handling P:C:I (25:24:1).Should a small spillage occur, outer gloves can then be disposed of without risk.
DO NOT leave tubes with P:C:I (25:24:1) for extended periods, it can degrade the plastic
?. [Extraction]
Transfer to the bead-beater and beat using the pre-programmed CTAB setting for . Return tubes to ice immediately after beating.
CTAB Bead beating Program:Speed: 5.5m/secAdapter: QuickprepTime: 60 secLysing Matrix: EQuantity: 1mlCycles: 1Pause time: 300sec
Ensure the lids of the LME tubes are securely fastened with no beads in the seal, and that the tubes are labelled on the top and on the side as the
?. [Extraction]
Centrifuge LME tubes at 16,000 x g for .
?. [Extraction]
Transfer all liquid to phase lock gel tube and keep the LME tube on ice.
Phase lock tubes should have been pre-spun. Phase lock gel should be pelleted at the bottom of each tube (often on the sides when they arrive from manufacturer). Centrifuge at 16,000 x g for
?. [Extraction]
Centrifuge the phase lock tube at 16,000 x g for at .
4 °C
Gel will form a barrier between the aqueous and P:C:I (25:24:1)/C:I (24:1) phases.
?. [Extraction]
Add 1 volume of C:I (24:1) to each phase lock gel tube, shake briefly to mix.  Centrifuge at 16,000 x g for  at .
4 °C
If barrier does not form, extend centrifugation time.
?. [Extraction]
Second extraction; In the fume hood, add  of Aluminium ammonium sulphate,  of CTAB extraction buffer and  of P:C:I (25:24:1) to each bead beating tube.
50 µl
500 µl
500 µl
?. [Extraction]
Repeat steps 8 to 13 then continue with the precipitation step below.
?. [Precipitation]
Transfer the aqueous phase from each tube to the pre-prepared eppendorfs with  of LPA in them.  As the solvent is locked beneath the phase lock gel, you should be able to pour this. You may need 2 eppendorfs per sample to fit allow room for PEG.
1 µl
?. [Precipitation]
Add 2 volumes of PEG/NaCl solution and mix well (solution is viscous).  Leave overnight in the fridge  at  to precipitate.
4 °C
If you are in a hurry it can be left for 2 hours but this will reduce the yield.
?. [Precipitation]
Centrifuge all eppendorfs at 16,000 x g for  at .
4 °C
?. [Precipitation]
Carefully aspirate the PEG/NaCl solution from the pellets.
Pellets are usually large and translucent.  They tend to be fairly uniform in size as the LPA has also precipitated.
?. [Precipitation]
Wash pellets with ice-cold 70 % ethanol to remove any precipitated salts and centrifuge at 16,000 x g for .
500 µl
?. [Precipitation]
Repeat the wash twice with  ice-cold 70 % ethanol.
200 µl
?. [Precipitation]
After briefly air drying the pellet for about 5 minutes, resuspend in low EDTA TE and combine the resuspended total nucleic acid from each sample into a single tube ( total per sample).  For sputum samples (or other high biomass samples) you may need to increase this volume to  per tube ( per sample).
30 µl
60 µl
50 µl
100 µl
We do not recommend storing DNA in cryo tubes as you are unable to spin them down.Recommended tubes:Tethered O ring, sterile tubes E1405-2142, star labs
?. [Precipitation]
This extract can be stored at  or    until required.
-20 °C
-80 °C
?. [Before you start]
Add working stock of Phenol:Chloroform:Isoamyl (P:C:I (25:24:1)) alcohol to a Falcon tubes in the fume cupboard.
50 mL
Use a 10ml pippette to remove required volume of P:C:I from below the the top buffered layer in the stock bottle. *For a new bottle ensure the buffer has been added and allowed to settle prior to starting the extraction.
?. [Before you start]
Add working stock of Chloroform:Isoamyl (C:I (24:1)) alcohol to a  Falcon tubes in the fume cupboard.
50 mL
?. [Before you start]
Pre-spin the 2 Phase-lock gel tubes ( Hard gel) per samples .Phase lock gel should be pelleted at the bottom of each tube (often on the sides when they arrive from manufacturer). Centrifuge at 16,000 x g for.
2 mL
?. [Before you start]
Add of linear polyacrylamide (LPA) to  eppendorfs, 2 per sample.This is a carrier and precipitates along with DNA increasing the yield. Unlike glycogen which can also be used it does not affect later sample use.
1 µl
2 mL