Lophelia Metagenome Extraction Protocol - Step 6

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Detailed Description

Video showing Step 5 of the  Lophelia Metagenome Extraction Protocol. 

6. DNA Extraction

The removal of the filter from the Sterivex cartridge is best done in a sterile environment like a laminar flow hood. After the filter is removed, the extraction is done using the Qiagen DNeasy extraction kit, following the Gram-positive bacteria protocol, but the reagent volumes are doubled in the initial steps [i.e., steps h to l below are steps 2 to 6 in the Qiagen Gram-positive protocol with volumes doubled; steps m - p are steps 4 to 7 of the Qiagen Animal Tissue protocol, using normal volumes]. Before beginning this step, make lysis buffer: 1- 2 samples, you will need to add 0.06g of powdered lysozyme to 3.24 ml of lysis buffer. Mix gently to dissolve or it will foam.

a. Remove Sterivex filter from 10 ml syringe. Place filter cartridge in sterile aluminum weigh dish.
b. Holding the input port of the Sterivex filter cartridge (the end the syringe was previously connected to), use flame-sterilized hammer to hit the seam at the opposite end of the cartridge to break the plastic outer case loose from the filter.
c. Use flame-sterilized forceps to pull the inner filter cartridge out and place it in a clean, sterile aluminum weigh dish.
d. Use clean, ethanol-sterilized razor blade to cut the filter on both sides of the visible seam.
e. Use flame-sterilized forceps to peel the filter off the cartridge (this may come off in one large piece or multiple smaller fragments).
f. Use ethanol-sterilized razor blade to cut filter into quarters (four roughly equal amounts).
g. Use flame-sterilized forceps to place each of the four filter fragments into a separate sterile 1.5 ml tube (total of four tubes per sample).
h. Add 360 vl of lysis buffer (after adding lysozyme) to each of the four tubes.
i. Incubate tubes at 37°C for 30 min. Vortex tubes every 10 min for 5 sec during this incubation to make sure filters are saturated.
j. Add 50 µl Proteinase K and 400 µl Buffer AL (without ethanol) to each of the four tubes. Mix by vortexing.
k. Incubate tubes at 56°C for 30 min. No vortexing.
l. Remove liquid from each of the four tubes to four new tubes. Pulse the old tubes in a microcentrifuge for 30 sec to release any remaining liquid. Transfer that liquid to new tubes also. Discard old tubes containing filter fragments. Add 400 µl of 99-100% ethanol to each tube and vortex. (Ethanol addition can cause a precipitate to form and if filters remained in the tubes, it could stick to them and be lost).
m. Pipet the mixture from each tube in the previous step to a DNeasy Mini spin column (one column per sample, so all four tubes will be combined onto one spin column). The spin column can hold ~ 750 µl per centrifugation, so it will take seven rounds of centrifugation to get all of the liquid onto the column. Centrifuge at 10,000xg for 1 min. Discard flow through liquid. Repeat until all liquid from the four tubes has been put through the column. Discard collection tube after final spin.
n. Place DNeasy Mini spin column in a new 2 ml collection tube (provided in kit), add 500 µl Buffer AW1 and centrifuge for 1 min at 10,000xg. Discard flow through and collection tube.
o. Place DNeasy Mini spin column in a new 2 ml collection tube (provided), add 500 µl Buffer AW2 and centrifuge for 3 min at 20,000xg (14,000 rpm) to dry the DNeasy membrane. Discard flow through and collection tube.
p. Place the DNeasy Mini spin column in a clean 1.5 ml tube and pipet 200 µl Buffer AE directly onto the DNeasy membrane. Incubate at room temperature for 1 min, and then centrifuge for 1 min at 10,000xg to elute. [The DNA can be frozen at this point until ready to proceed.] 

Details

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Location Taken: US

Transcript

6. DNA Extraction

The removal of the filter from the Sterivex cartridge is best done in a sterile environment like a laminar flow hood. After the filter is removed, the extraction is done using the Qiagen DNeasy extraction kit, following the Gram-positive bacteria protocol, but the reagent volumes are doubled in the initial steps [i.e., steps h to l below are steps 2 to 6 in the Qiagen Gram-positive protocol with volumes doubled; steps m - p are steps 4 to 7 of the Qiagen Animal Tissue protocol, using normal volumes]. Before beginning this step, make lysis buffer: 1- 2 samples, you will need to add 0.06g of powdered lysozyme to 3.24 ml of lysis buffer. Mix gently to dissolve or it will foam.

a. Remove Sterivex filter from 10 ml syringe. Place filter cartridge in sterile aluminum weigh dish.
b. Holding the input port of the Sterivex filter cartridge (the end the syringe was previously connected to), use flame-sterilized hammer to hit the seam at the opposite end of the cartridge to break the plastic outer case loose from the filter.
c. Use flame-sterilized forceps to pull the inner filter cartridge out and place it in a clean, sterile aluminum weigh dish.
d. Use clean, ethanol-sterilized razor blade to cut the filter on both sides of the visible seam.
e. Use flame-sterilized forceps to peel the filter off the cartridge (this may come off in one large piece or multiple smaller fragments).
f. Use ethanol-sterilized razor blade to cut filter into quarters (four roughly equal amounts).
g. Use flame-sterilized forceps to place each of the four filter fragments into a separate sterile 1.5 ml tube (total of four tubes per sample).
h. Add 360 vl of lysis buffer (after adding lysozyme) to each of the four tubes.
i. Incubate tubes at 37°C for 30 min. Vortex tubes every 10 min for 5 sec during this incubation to make sure filters are saturated.
j. Add 50 µl Proteinase K and 400 µl Buffer AL (without ethanol) to each of the four tubes. Mix by vortexing.
k. Incubate tubes at 56°C for 30 min. No vortexing.
l. Remove liquid from each of the four tubes to four new tubes. Pulse the old tubes in a microcentrifuge for 30 sec to release any remaining liquid. Transfer that liquid to new tubes also. Discard old tubes containing filter fragments. Add 400 µl of 99-100% ethanol to each tube and vortex. (Ethanol addition can cause a precipitate to form and if filters remained in the tubes, it could stick to them and be lost).
m. Pipet the mixture from each tube in the previous step to a DNeasy Mini spin column (one column per sample, so all four tubes will be combined onto one spin column). The spin column can hold ~ 750 µl per centrifugation, so it will take seven rounds of centrifugation to get all of the liquid onto the column. Centrifuge at 10,000xg for 1 min. Discard flow through liquid. Repeat until all liquid from the four tubes has been put through the column. Discard collection tube after final spin.
n. Place DNeasy Mini spin column in a new 2 ml collection tube (provided in kit), add 500 µl Buffer AW1 and centrifuge for 1 min at 10,000xg. Discard flow through and collection tube.
o. Place DNeasy Mini spin column in a new 2 ml collection tube (provided), add 500 µl Buffer AW2 and centrifuge for 3 min at 20,000xg (14,000 rpm) to dry the DNeasy membrane. Discard flow through and collection tube.
p. Place the DNeasy Mini spin column in a clean 1.5 ml tube and pipet 200 µl Buffer AE directly onto the DNeasy membrane. Incubate at room temperature for 1 min, and then centrifuge for 1 min at 10,000xg to elute. [The DNA can be frozen at this point until ready to proceed.]