diff --git a/docs/_Experimental-Procedure-Standards-SOPs/00_sop-template b/docs/_Experimental-Procedure-Standards-SOPs/00_sop-template new file mode 100644 index 0000000..42b86cf --- /dev/null +++ b/docs/_Experimental-Procedure-Standards-SOPs/00_sop-template @@ -0,0 +1,54 @@ +--- +title: Template for SOP +category: Experimental-Procedure-Standards-SOPs +layout: default +docs_css: markdown +hide: true +--- + +This is a template document on how to add templates to the knowledge base. Please create a copy and adapt accordingly. + + +# Metadata + +| Title | Category | Adapated and used by | last accessed | Link to protocol | Primary origin for protocol | +| ------ | ------ | ------ | ------ | ------ |------ | +| Media for freezing homogenised faecal samples for re-cultivation / FMT | Media preparation | University Hospital of RWTH Aachen, Inst. Med. Microbiology, Clavel lab +Collaborative Research Center 1382, Project Q02 “Integrative Microbiota Analysis”, Dr. Nicole Treichel | 2024-09-12 | {% cite fmt_media %} | {% cite burz_2019 %} | +{: .table .table-hover} + + +# add references to the bib file add: +Here are a few examples: + +@misc{fmt_media, + title = {Media for freezing homogenised faecal samples for re-cultivation / FMT}, + author = {Treichel, Nicole}, + publisher = {CRC1382}, + howpublished = {\url{https://www.crc1382.org/wp-content/uploads/2022/11/221019_FMT-Media-Protocol.pdf}}, + urldate = {2024-09-12}, + year = {2022}, + month = {Nov} +} + +@article{burz_2019, + author = {Burz, S.D., Abraham, AL., Fonseca, F. et al.}, + title = {A Guide for Ex Vivo Handling and Storage of Stool Samples Intended for Fecal Microbiota Transplantation.}, + doi = {10.1038/s41598-019-45173-4}, + journal = {Scientific Reports}, + year = {2019}, + month = {06}, + volume = {9}, + url = {https://doi.org/10.1038/s41598-019-45173-4} +} + +# Protocol + +Add your protocol here. + +##Method + +##Material + +# References +{% bibliography --cited_in_order %} diff --git a/docs/_Experimental-Procedure-Standards-SOPs/09-sample-collection-and-storage-by-sample.md b/docs/_Experimental-Procedure-Standards-SOPs/09-sample-collection-and-storage-by-sample.md index be6a62c..86df6c0 100644 --- a/docs/_Experimental-Procedure-Standards-SOPs/09-sample-collection-and-storage-by-sample.md +++ b/docs/_Experimental-Procedure-Standards-SOPs/09-sample-collection-and-storage-by-sample.md @@ -13,34 +13,34 @@ Protein storage at -20°C usually requires the addition of 50% glycerol to your Protein samples stored at -80ºC will be frozen. As repeated freeze-thaw cycles usually have a negative influence on protein samples, it is best to prepare small-sized, single-use aliquots that will be used up during the course of an experiment. 5-10% glycerol or other additives that protect against the effect of freezing and thawing can be added as well. After preparing your protein sample aliquots, it is important to flash-freeze them in liquid nitrogen before importing them into the -80ºC freezer for long-term storage. Many proteins are stable for months to years when stored under appropriate conditions at -80°C, but the exact time frame varies from protein to protein and should be determined experimentally. -| Sample | Procedure | Source or Link | -|---------|------------|-----------------| -| Activated sludge from wastewater | collected samples stored at 4°C for transport, within 6 h centrifuged at 6 500 g for 15 min at 4°C, supernatant removed and pellets stored at -20°C | {% cite Morin_2020 %} | -| Antarctic ticks | collected from rocks, placed in RNALater and stored at -80°C within 4-8 h of collection | {% cite Wille_2020 %} | -| Atlantic salmon | dissection samples placed in RNAlater and stored at -80°C | {% cite Karlsen_2022 %} | -| Faeces/gut content | flash freeze in liquid nitrogen and store at -80°C (dissolve in Stool DNA Stabilizer (INVITEK Molecular) while thawing; or if you have more time dissolve in Stool DNA Stabilizer right after collection and normally freeze and store at -80°C) | (in-house protocol provided by Johannes Masson) | -| Frog swabbing | rinse caught frog with 50 ml sterilised water to remove transient bacteria, swab entire skin surface with a sterile Dacron swab for 30 sec, place swab in microcentrifuge tube containing PBS and store on ice, then transfer to -80°C storage | {% cite Ellison_2021 %} | -| Frog toes | stored in sterile 1.5 ml tubes at -80°C | {% cite Boyle_2004 %} | -| Fruit fly larvae | sterilise larvae with a solution of 0.5 % Tween 80, 0.5 % sodium hypochlorite and 80 % ethanol for 30 sec, rinse them 3 times in PBS for 30 sec, use sterile pestle to crush larvae and store in BHI containing 20 % glycerol at -80°C | {% cite Majumder_2020 %} | -| Hooded seal brain | whole samples are placed in 4°C glucose artificial cerebrospinal fluid (aCSF) saturated with 95 % O2 - 5 % CO2. After experiment (induced normoxia and hypoxia), tissues were stored in RNAlater stabilisation fluid (Life Technologies, USA) and kept at 4°C. | (in-house protocol provided by Justine Vandedorpe) | -| Invertebrates | whole samples are stored in 80 % ethanol, in fridge for short-term storage, and in -20°C freezer for long-term storage | (in-house protocol provided by Justine Vandedorpe) | -| Leaves | surface sterilise: rinse for 5 sec 95% ethanol, 30 sec in 70% ethanol, 30 sec in 10% bleach, followed by three 2-minute washes in sterile water, air-dry for 5 min, then add to 2 ml tube containing 350 µL 10 mM MgSO4 and a sterile 5 mm steel ball and homogenise using a TissueLyser (QIAGEN) run at max speed (50 Hz) for up to 60 sec, then flash freeze in liquid nitrogen and store at -80°C | {% cite Humphrey_2020 %} | -| Maternal Milk (with glycerol) | Minimum of 2ml of milk was self-collected by the mothers in sterile falcon tubes with added 20% glycerol while wearing disposable gloves. Samples without were stored at -20C for cultivation experiments. Note that for colostrum and other milk samples within the first week of life the 2ml min quantity might not be possible. In that case, the mother collected whatever quantity was possible in 10 min timespan. | (in-house protocol provided by Pamela Ferretti) | -| Maternal Milk (without glycerol) | Minimum of 2ml of milk was self-collected by the mothers in sterile falcon tubes while wearing disposable gloves. No buffer was added. Samples were stored at -4C right after collection and moved to -80C within a week. Note that for colostrum and other milk samples within the first week of life the 2ml min quantity might not be possible. In that case, the mother collected whatever quantity was possible in 10 min timespan. | (in-house protocol provided by Pamela Ferretti) | -| Mosquitos | human bait adult catching method, pool 3-5 mosquitos, store at -80°C | {% cite Ali_2021 %} | -| Oral swabs | after collection swab is immersed in 10:1 Tris-EDTA buffer immediately and stored at −80 °C | {% cite Gao_2020 %} | -| Penguin cloacal swaps | placed in tubes containing viral transport medium (brain heart infusion [BHI] broth-based medium [Oxoid] with 0.3 mg/ml penicillin, 5 mg/ml streptomycin, 0.1 mg/ml gentamicin, and 2.5 μg/ml amphotericin B), kept on ice for 4 hours, then stored at -80°C | {% cite Wille_2020 %} | -| Pooled tear collection | centrifuged at 270 g for 3 min, supernatant stored in 1.5 ml microcentrifuge tubes at -80°C | {% cite Willis_2020 %} | -| Prokaryotes in sea water | water sampled without filtration, 2% final concentration glutaraldehyde added, stored at 4°C for 15 min in the dark, flash-frozen in liquid nitrogen and stored at -80°C | {% cite Baltar_2018 %} | -| Saliva | passive drooling procedure in sterile 50 ml screw top tubes (approximately 3 ml of saliva per sample). About 30 minutes before collection, all volunteers performed a mouth rinse with drinking water. Samples were vortexed before 2 ml of each was centrifuged at 10,000 × g for 10 minutes to collected the bacterial pellets. Total DNA was extracted using the PowerSoil DNA Isolation Kit (MoBio), according to manufacturer’s instructions. DNA was stored at −20 °C until used. | (in-house protocol provided by Pamela Ferretti) | -| Seawater | filtered through a 200 µm, then a 20 µm mesh, then vacuum filtered through a 0.8 µm filter, then a 0.2 µm filter, flash frozen in liquid nitrogen & stored at -80°C (all done with ~4h; water at 4°C during processing) | {% cite Acinas_2021 %} | -| Seawater | filtered through a 0.2 µm filter, filter stored in 2 ml tubes containing lysis buffer (EDTA 40 mmol l−1 pH 8.0, Tris-HCl 50 mmol l−1 pH 8.0, sucrose 0.75 mol l−1) flash frozen in liquid nitrogen, filtered & unfiltered water stored in 15 ml falcons at -20°C | {% cite Michoud_2021 %} | +| Sample | Procedure | Source or Link | +|----------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------| +| Activated sludge from wastewater | collected samples stored at 4°C for transport, within 6 h centrifuged at 6 500 g for 15 min at 4°C, supernatant removed and pellets stored at -20°C | {% cite Morin_2020 %} | +| Antarctic ticks | collected from rocks, placed in RNALater and stored at -80°C within 4-8 h of collection | {% cite Wille_2020 %} | +| Atlantic salmon | dissection samples placed in RNAlater and stored at -80°C | {% cite Karlsen_2022 %} | +| Faeces/gut content | flash freeze in liquid nitrogen and store at -80°C (dissolve in Stool DNA Stabilizer (INVITEK Molecular) while thawing; or if you have more time dissolve in Stool DNA Stabilizer right after collection and normally freeze and store at -80°C) | (in-house protocol provided by Johannes Masson) | +| Frog swabbing | rinse caught frog with 50 ml sterilised water to remove transient bacteria, swab entire skin surface with a sterile Dacron swab for 30 sec, place swab in microcentrifuge tube containing PBS and store on ice, then transfer to -80°C storage | {% cite Ellison_2021 %} | +| Frog toes | stored in sterile 1.5 ml tubes at -80°C | {% cite Boyle_2004 %} | +| Fruit fly larvae | sterilise larvae with a solution of 0.5 % Tween 80, 0.5 % sodium hypochlorite and 80 % ethanol for 30 sec, rinse them 3 times in PBS for 30 sec, use sterile pestle to crush larvae and store in BHI containing 20 % glycerol at -80°C | {% cite Majumder_2020 %} | +| Hooded seal brain | whole samples are placed in 4°C glucose artificial cerebrospinal fluid (aCSF) saturated with 95 % O2 - 5 % CO2. After experiment (induced normoxia and hypoxia), tissues were stored in RNAlater stabilisation fluid (Life Technologies, USA) and kept at 4°C.| (in-house protocol provided by Justine Vandedorpe) | +| Invertebrates | whole samples are stored in 80 % ethanol, in fridge for short-term storage, and in -20°C freezer for long-term storage | (in-house protocol provided by Justine Vandedorpe) | +| Leaves | surface sterilise: rinse for 5 sec 95% ethanol, 30 sec in 70% ethanol, 30 sec in 10% bleach, followed by three 2-minute washes in sterile water, air-dry for 5 min, then add to 2 ml tube containing 350 µL 10 mM MgSO4 and a sterile 5 mm steel ball and homogenise using a TissueLyser (QIAGEN) run at max speed (50 Hz) for up to 60 sec, then flash freeze in liquid nitrogen and store at -80°C | {% cite Humphrey_2020 %} | +| Maternal Milk (with glycerol) | Minimum of 2ml of milk was self-collected by the mothers in sterile falcon tubes with added 20% glycerol while wearing disposable gloves. Samples without were stored at -20C for cultivation experiments. Note that for colostrum and other milk samples within the first week of life the 2ml min quantity might not be possible. In that case, the mother collected whatever quantity was possible in 10 min timespan. | (in-house protocol provided by Pamela Ferretti) | +| Maternal Milk (without glycerol) | Minimum of 2ml of milk was self-collected by the mothers in sterile falcon tubes while wearing disposable gloves. No buffer was added. Samples were stored at -4C right after collection and moved to -80C within a week. Note that for colostrum and other milk samples within the first week of life the 2ml min quantity might not be possible. In that case, the mother collected whatever quantity was possible in 10 min timespan. | (in-house protocol provided by Pamela Ferretti) | +| Mosquitos | human bait adult catching method, pool 3-5 mosquitos, store at -80°C | {% cite Ali_2021 %} | +| Oral swabs | after collection swab is immersed in 10:1 Tris-EDTA buffer immediately and stored at −80 °C | {% cite Gao_2020 %} | +| Penguin cloacal swaps | placed in tubes containing viral transport medium (brain heart infusion [BHI] broth-based medium [Oxoid] with 0.3 mg/ml penicillin, 5 mg/ml streptomycin, 0.1 mg/ml gentamicin, and 2.5 μg/ml amphotericin B), kept on ice for 4 hours, then stored at -80°C | {% cite Wille_2020 %} | +| Pooled tear collection | centrifuged at 270 g for 3 min, supernatant stored in 1.5 ml microcentrifuge tubes at -80°C | {% cite Willis_2020 %} | +| Prokaryotes in sea water | water sampled without filtration, 2% final concentration glutaraldehyde added, stored at 4°C for 15 min in the dark, flash-frozen in liquid nitrogen and stored at -80°C | {% cite Baltar_2018 %} | +| Saliva | passive drooling procedure in sterile 50 ml screw top tubes (approximately 3 ml of saliva per sample). About 30 minutes before collection, all volunteers performed a mouth rinse with drinking water. Samples were vortexed before 2 ml of each was centrifuged at 10,000 × g for 10 minutes to collected the bacterial pellets. Total DNA was extracted using the PowerSoil DNA Isolation Kit (MoBio), according to manufacturer’s instructions. DNA was stored at −20 °C until used. | (in-house protocol provided by Pamela Ferretti) | +| Seawater | filtered through a 200 µm, then a 20 µm mesh, then vacuum filtered through a 0.8 µm filter, then a 0.2 µm filter, flash frozen in liquid nitrogen & stored at -80°C (all done with ~4h; water at 4°C during processing) | {% cite Acinas_2021 %} | +| Seawater | filtered through a 0.2 µm filter, filter stored in 2 ml tubes containing lysis buffer (EDTA 40 mmol l−1 pH 8.0, Tris-HCl 50 mmol l−1 pH 8.0, sucrose 0.75 mol l−1) flash frozen in liquid nitrogen, filtered & unfiltered water stored in 15 ml falcons at -20°C | {% cite Michoud_2021 %} | | Skin swab | Skin samples were collected using Catch-All-Swabs (Epicentre Technologies, Wisconsin, USA) shortly after birth but before the skin-to-skin contact with the infant, by swabbing the upper area of the maternal breast (intermammary cleft). After pre-moistening with 2 ml SCF-1 buffer (50 mM Tris buffer, pH 7.6, 1mM EDTA, pH 8.0, and 0.5% Tween-20) (Human Microbiome Project Consortium, 2012) contained in a 15 ml sterile screw top collection tube (Sarstedt, Nümbrecht, Germany), the swab head was rubbed back and forth for approximately 30 seconds over the area (repeating twice) before the swab was returned to the buffered solution. The lower part of the swab was broken to ensure closure of the tube (see below). After sampling, deterge the sampled area with a clean pad of cotton and water. | (in-house protocol provided by Pamela Ferretti) | -| Sponges | washed three times with filter-sterilised natural seawater, one fragment placed in 70% ethanol, one fragment placed in RNALater, stored at -20°C | {% cite Ruocco_2021 %} | -| Stool samples | samples were self-collected using collection tubes specific for faecal material (Sarstedt, Nümbrecht, Germany). Toilet paper was placed at the bottom of the WC, to prevent stool samples from sinking and getting contaminated. The collection was performed at the upper part of the feces, the one not in contact with the toilet paper, WC walls or other material. Subjects were instructed, whenever possible, to urinate before evacuating the stools. Samples were collected in variable quantity, depending on availability (meconium is usually only one spoon). Once collected the tube was placed at -4C as soon as possible, and then transferred to -80C within a week. | (in-house protocol provided by Pamela Ferretti) | -| Swedish cervical biobank | stored in liquid at -25°C, using liquid-based cytology cells suspended and fixed in Thinprep (TP) containing 20 ml PreservCyt (ThinPrep Hologic, Boxborough, MA). 4 ml of liquid-based cytology sample from the bottom of the patient tube is transferred into a conical tube and allowed to sediment for 30 min. 300 µl of the sediment are transferred to a cryotube and stored at -25°C | {% cite Perskvist_2013 %} | -| Tongue dorsum swabs | Skin samples were collected using Catch-All-Swabs (Epicentre Technologies, Wisconsin, USA). Samples were collected by rubbing a swab on the central area of the back of the tongue for approximately 5 seconds. The swab head was then placed in a 15 ml collection tube containing 2 ml SCF-1 buffer (see photos above). Samples were collected while wearing protective disposable gloves to avoid skin contamination. | (in-house protocol provided by Pamela Ferretti) | -| Turkey Trachea swabs | placed in 1 ml PBS, then stored at -20°C | {% cite Kursa_2021 %} | -| Vaginal swabs | Skin samples were collected using Catch-All-Swabs (Epicentre Technologies, Wisconsin, USA). The swab was rubbed 5 times, with a circular motion, in the vaginal introitus and then the swab head was placed in a 15 ml sterile screw top collection tube containing 2 ml SCF-1 buffer (see photos above). | (in-house protocol provided by Pamela Ferretti) | -| Yoghurt | after purchase, samples were placed immediately at 4°C for transport and within 12 h stared at -80°C | {% cite Islam_2021 %} | +| Sponges | washed three times with filter-sterilised natural seawater, one fragment placed in 70% ethanol, one fragment placed in RNALater, stored at -20°C | {% cite Ruocco_2021 %} | +| Stool samples | samples were self-collected using collection tubes specific for faecal material (Sarstedt, Nümbrecht, Germany). Toilet paper was placed at the bottom of the WC, to prevent stool samples from sinking and getting contaminated. The collection was performed at the upper part of the feces, the one not in contact with the toilet paper, WC walls or other material. Subjects were instructed, whenever possible, to urinate before evacuating the stools. Samples were collected in variable quantity, depending on availability (meconium is usually only one spoon). Once collected the tube was placed at -4C as soon as possible, and then transferred to -80C within a week. | (in-house protocol provided by Pamela Ferretti) | +| Swedish cervical biobank | stored in liquid at -25°C, using liquid-based cytology cells suspended and fixed in Thinprep (TP) containing 20 ml PreservCyt (ThinPrep Hologic, Boxborough, MA). 4 ml of liquid-based cytology sample from the bottom of the patient tube is transferred into a conical tube and allowed to sediment for 30 min. 300 µl of the sediment are transferred to a cryotube and stored at -25°C | {% cite Perskvist_2013 %} | +| Tongue dorsum swabs | Skin samples were collected using Catch-All-Swabs (Epicentre Technologies, Wisconsin, USA). Samples were collected by rubbing a swab on the central area of the back of the tongue for approximately 5 seconds. The swab head was then placed in a 15 ml collection tube containing 2 ml SCF-1 buffer (see photos above). Samples were collected while wearing protective disposable gloves to avoid skin contamination. | (in-house protocol provided by Pamela Ferretti) | +| Turkey Trachea swabs | placed in 1 ml PBS, then stored at -20°C | {% cite Kursa_2021 %} | +| Vaginal swabs | Skin samples were collected using Catch-All-Swabs (Epicentre Technologies, Wisconsin, USA). The swab was rubbed 5 times, with a circular motion, in the vaginal introitus and then the swab head was placed in a 15 ml sterile screw top collection tube containing 2 ml SCF-1 buffer (see photos above). | (in-house protocol provided by Pamela Ferretti) | +| Yoghurt | after purchase, samples were placed immediately at 4°C for transport and within 12 h stared at -80°C | {% cite Islam_2021 %}| {: .table .table-hover} \ No newline at end of file diff --git a/docs/_Experimental-Procedure-Standards-SOPs/10-media-freezing-faecal-samples-recultivation b/docs/_Experimental-Procedure-Standards-SOPs/10-media-freezing-faecal-samples-recultivation new file mode 100644 index 0000000..d500e4c --- /dev/null +++ b/docs/_Experimental-Procedure-Standards-SOPs/10-media-freezing-faecal-samples-recultivation @@ -0,0 +1,45 @@ +--- +title: Media for freezing homogenised faecal samples for re-cultivation +category: Experimental-Procedure-Standards-SOPs +layout: default +docs_css: markdown +hide: false +--- + +# Metadata + +| Title | Adapated and used by | last accessed | Link to protocol | Primary origin for protocol | +| ------ | ------ | ------ | ------ | ------ | +| Media for freezing homogenised faecal samples for re-cultivation / FMT | University Hospital of RWTH Aachen, Inst. Med. Microbiology, Clavel lab +Collaborative Research Center 1382, Project Q02 “Integrative Microbiota Analysis”, Dr. Nicole Treichel | 2024-09-12 | {% cite fmt_media %} | {% cite burz_2019 %} | +{: .table .table-hover} + + +# Protocol + + +1. Make normal saline: 9.0 g/L NaCl +2. Dissolve Maltodextrin and Trehalose in normal saline at 40 °C, to respective final concentrations +of 18.75% and 6.25% (w/v; 1% = 1 g/100 mL). Add 0.5% (w/v) Ascorbic acid and 0.05% L-Cystein +(w/v). + + +For 100 mL: +|amount | ingredient | +18.75 g | Maltodextrin | +6.25 g | Trehalose | +0.5 g | Ascorbic acid | +0.05 g | L-Cystein | + +## Material +- Maltodextrin [PubChem SID: 481109074](https://pubchem.ncbi.nlm.nih.gov/substance/481109074) +- Trehalose +- L-Cystein +- Ascorbic acid +- NaCl +- Water (MQ/DEPC) + + +# References +{% bibliography --cited_in_order %} + diff --git a/docs/_Experimental-Procedure-Standards-SOPs/11-biobank-fmt-samples b/docs/_Experimental-Procedure-Standards-SOPs/11-biobank-fmt-samples new file mode 100644 index 0000000..ebf15b8 --- /dev/null +++ b/docs/_Experimental-Procedure-Standards-SOPs/11-biobank-fmt-samples @@ -0,0 +1,79 @@ +--- +title: Biobank of human stool samples – sample processing +category: Experimental-Procedure-Standards-SOPs +layout: default +docs_css: markdown +hide: true +--- + +# Metadata + +| Title | Category | Adapated and used by | last accessed | Link to protocol | Primary origin for protocol | +| ------ | ------ | ------ | ------ | ------ |-------| +| Biobank of human stool samples – sample processing | Sample processing | University Hospital of RWTH Aachen, Inst. Med. Microbiology, Clavel lab +Collaborative Research Center 1382, Project Q02 “Integrative Microbiota Analysis”, Dr. Nicole Treichel | 2024-09-12 | {% cite fmt_biobank %} | N/A | +{: .table .table-hover} + + +# Protocol + +For each sample (001-NNN), label 50 x 1,5 mL Eppendorf Tubes as follows (example for project acronym +“Cultimic”): +C Cultimic-001 15x (Cultivation) +S Cultimic-001 5x (Sequencing) +U Cultimic-001 5x (Untreated) +F Cultimic-001 20x (FACS) +M Cultimic-001 5x (Metabolomics) + +Work hereon under the fume hood due to smell. +Take the stool sample in flexible plastic bag out of the plastic container and homogenise by kneading the +bag manually (be careful to not let air inside). +1. **Cultivation**: In a 50 mL Falcon tube, mix thoroughly (vortexing) approx. 3 g of stool with 12 mL of +FMT medium; let larger debris set for 1 min; distribute into 15 x C-tubes, 1 mL each; use pipet tips +with a wide opening to avoid clogging. (Alternatively, cut the end of 1000µL tips with a sterile +scissor) +[Store 100µL of FMT medium as such to serve as negative control]. + + +2. **Sequencing**: mix approx. 1 g of stool with 6 mL DNA stabilizer; homogenize by vortexing and +distribute into 5 x S-tubes, 1 mL each; use tips with a wide opening to avoid clogging. +[Store 600µL of DNA stabilizer as such to serve as negative control]. + + +3. **Untreated** (for any types of analysis for which additional reagents confound the results): With a +sterile spatula, fill 5 x U-tubes with ca. 600 mg of untreated stool sample. + + +4. **Flow Cytometry** (equivalent to untreated): With a spatula, fill 20 x F-tubes with ca. 100 mg of +untreated stool sample. + + +5. **Metabolomics (faecal water)**: Weigh a 50 mL falcon tube. Fill it with approx. 1 g of stool. Weigh the +tube again and calculate the difference to determine the exact amount of stool. Dilute with 4 mL of +water (molecular biology grade, DEPC treated, and filter sterilised). Distribute into 2 mL Eppendorf +Tubes and centrifuge (12,000 x g, 3 min). Filter the supernatants using a syringe fitted with a 0.20 +µm filter and distribute into 5 x M-tubes. Protect yourself (e.g. wear safety googles) during the +filtration step, as the filter may disconnect abruptly from the syringe and faecal water be spilled. +[Store 2 negative controls: water only, and filtered water]. + + +Transfer all samples to -80°C freezer immediately after preparation. +Collect questionnaires and enter metadata into the protected, study-specific spreadsheet. + + +##Material +- Stomacher® 400 Classic Bags- Standard Bag Irradiated Sterile; VWR (BA6041/5) +- BD GasPak™ EZ Anaerobe Pouch System with indicator; Becton Dickinson (260683) +- Probengefäße White 1000 ml 130 mm(80), VWR (216-4278) +- Nitril Gloves +- Stool DNA Stabilizer; invitek molecular (1038111100) +- Eppendorf tubes 1.5 mL and 2 mL +- Water for molecular biology, DEPC-treated and filter-sterilised +- FMT medium (see separate protocol) +- 0.20-µm sterile syringe filter; Corning inc. (431219) +- 1 mL syringes +- 50 mL Falcon tubes +- Pipet tips with wide opening 1250µL; VWR (613-0751) + +# References +{% bibliography --cited_in_order %} diff --git a/docs/_Experimental-Procedure-Standards-SOPs/12-godon-dna-extraction b/docs/_Experimental-Procedure-Standards-SOPs/12-godon-dna-extraction new file mode 100644 index 0000000..01a49ca --- /dev/null +++ b/docs/_Experimental-Procedure-Standards-SOPs/12-godon-dna-extraction @@ -0,0 +1,83 @@ +--- +title: DNA extraction: Shortened Godon protocol with optional enzymatic lysis +category: Experimental-Procedure-Standards-SOPs +layout: default +docs_css: markdown +hide: true +--- + + +# Metadata + +| Title | Category | Adapated and used by | last accessed | Link to protocol | Primary origin for protocol | +| ------ | ------ | ------ | ------ | ------ |------ | +| DNA extraction: Shortened Godon protocol with optional enzymatic lysis | DNA extraction | University Hospital of RWTH Aachen, Inst. Med. Microbiology, Clavel lab +Collaborative Research Center 1382, Project Q02 “Integrative Microbiota Analysis”, Dr. Nicole Treichel | 2024-09-12 | {% cite godon_dna_extraction %} | {% cite godon_1997 %} | +{: .table .table-hover} + +*Consider accessing the protocol for a donwloadable PDF version.* + +# Protocol + +## Prepare: +- TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) +- Bead-beater-tubes (with 500 mg+/- 10 mg Zirconia/Silicia beads, autoclaved) +- 4M Guanidinethiocyanate in 0,1M Tris pH 7,5 (consider appropriate disposal) +- 5 % N-laurolylsarcosine in PBS (Dulbecco’s phosphate Buffered Saline) (consider appropriate disposal) + + +## Sample preparation: +Consider first if you want to use the enzymatic lysis step! +- For extraction from bacterial culture: Take 2 ml of a well grown culture, centrifuge it at 12.000 x g for 10 min, and discard +the supernatant +- For faecal samples stored premixed 1:6 with DNA Stabilizer (recommended for protocol used without enzymatic step): let +thaw at room temperature. +- For faecal samples stored without DNA Stabilizer, add DNA stabilizer (1:6) to frozen sample, mix and thaw at room +temperature +- If protocol is used with enzymatic step do not use DNA Stabilizer + +## DNA Isolation: +**For enzymatic extraction** +Prepare Lysozyme-Solution (TE Buffer with 15 µg/µl Lysozyme): + mass Lysozyme = (#Samples(+1)) * 7.5 mg + Volume TE buffer = (#Samples(+1)) * 0.5 ml +Weigh lysozyme into TE buffer (leave lysozyme stock on ice!) +1. Resuspend bacterial pellet or faecal sample in 500 µl lysozyme-solution +2. Incubate for 30 min at 37 °C +3. Add 50 µl 10 % SDS +4. Add 15 µl ProteinaseK (20 mg/ml) +5. Incubate for 1-2 h at 50 °C (liquid should be totally clear) +Continue with step 2 below + +**Without enzymatic extraction** +For extraction without the enzymatic step, resuspend the bacterial pellet in 600 µl DNA Stool Stabilizer or use +600µL of faecal sample in DNA stabilizer +2. Transfer sample into bead-beater-tubes (with 500 mg+/- 10 mg Zirconia/Silicia beads, previously autoclaved) +3. Add 250 µl 4M Guanidinethiocyanate +4. Add 500 µl 5 % N-laurolylsarcosine  vortex +5. Incubate at 70 °C while shaking (700 rpm) for 60 min; Set centrifuge to 4 °C +6. Bead-beat 3 x with the following program; re-fill the cooling adapter of the bead-beater with dry ice between each +round: CY; 40 s; 6,6 m/s, cooled with dry ice +7. Add 15 mg PVPP [Poly(vinylpolypyrrolidone)] +Vortex, then centrifuge at 15.000 x g, 4 °C, 3 min +8. Transfer clear supernatant into a new 2 ml tube +Centrifuge at 15.000 x g, 4 °C, 3 min +9. Take 500 µl clear supernatant into a new 2 ml tube +10. Add 5 µl RNase (10 mg/ml), incubate at 37 °C while shaking (700 rpm) for 20-30 min (heat incubator afterwards to 70 °C, +heat up DE) +11. Proceed with the NucleoSpin® gDNA Clean-up follow protocol + +##Material + +- ProteinaseK; Carl Roth (7528.1) +- NucleoSpin® gDNA Clean-up; Macherey-Nagel (740230.250) +- RNase; Sigma-Aldrich (R6513-50mg) +- Guanidinethiocyanate; Sigma-Aldrich (G9277-100G) +- Zirconia/Silicia beads; Biozym (55D1132-01TP) +- N-laurolylsarcosine; Sigma-Aldrich (61743-25G) +- TE buffer; Sigma-Aldrich (T9285-100ML) +- Poly(vinylpolypyrrolidone); Sigma-Aldrich (P5288-100G) +- 1.5 mL and 2 mL Eppendorf tubes + +# References +{% bibliography --cited_in_order %} diff --git a/docs/_bibliography/references.bib b/docs/_bibliography/references.bib index 696b66d..fe9412a 100644 --- a/docs/_bibliography/references.bib +++ b/docs/_bibliography/references.bib @@ -767,6 +767,29 @@ @misc{FDA:2021 year = {2021} } + +@misc{fmt_media, + title = {Media for freezing homogenised faecal samples for re-cultivation / FMT}, + author = {Treichel, Nicole}, + publisher = {CRC1382}, + howpublished = {\url{https://www.crc1382.org/wp-content/uploads/2022/11/221019_FMT-Media-Protocol.pdf}}, + urldate = {2024-09-12}, + year = {2022}, + month = {Nov} +} + +@article{burz_2019, + author = {Burz, S.D., Abraham, AL., Fonseca, F. et al.}, + title = {A Guide for Ex Vivo Handling and Storage of Stool Samples Intended for Fecal Microbiota Transplantation.}, + doi = {10.1038/s41598-019-45173-4}, + journal = {Scientific Reports}, + year = {2019}, + month = {06}, + volume = {9}, + url = {https://doi.org/10.1038/s41598-019-45173-4} + } + + @MISC{Adam:2021, title = "{ELN} Guide", author = "Adam, @@ -949,3 +972,35 @@ @book{CCSDS_OAIS:2012 author = {{Consultative Committee for Space Data Systems CCSDS}}, year = {2012}, } + +@misc{fmt_biobank, + title = {Biobank of human stool samples – sample processing}, + author = {Treichel, Nicole}, + publisher = {CRC1382}, + howpublished = {\url{https://www.crc1382.org/wp-content/uploads/2022/11/221019_Biobank-of-human-stool-samples-sample-processing.pdf}}, + urldate = {2024-09-12}, + year = {2022}, + month = {09} +} + +@misc{godon_dna_extraction, + title = {DNA extraction: Shortened Godon protocol with optional enzymatic lysis}, + author = {Treichel, Nicole}, + publisher = {CRC1382}, + howpublished = {\url{https://www.crc1382.org/wp-content/uploads/2022/11/221019_DNA-extraction.pdf}}, + urldate = {2024-09-12}, + year = {2022}, + month = {09} +} + +@article{godon_1997, + author = {Godon JJ, Zumstein E, Dabert P, Habouzit F, Moletta R.}, + title = {Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA +sequence analysis}, + doi = {10.1128/aem.63.7.2802-2813.1997}, + journal = {Appl Environ Microbiol.}, + year = {1997}, + month = {07}, + volume = {63}, + url = {https://journals.asm.org/doi/10.1128/aem.63.7.2802-2813.1997} +}