Name: Large-Scale Screens of Metagenomic Libraries (Video) | JoVE Journal
Uploaded: 2007-05-28T00:00:00.000+00:00
Duration: 16 min 5 s
Description: Learn robotic inoculation of metagenomic libraries onto membrane filters for high-throughput genomics. This method enables accurate placement of 10,000 colonies per filter, advancing microbial community research.

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5 minutes

1. Hybridization procedure
One hybridization tube will take 1 or 2 membranes (size 22 by 22 cm). Use 50 ml of hybridization mixture and 0.5 mg of DNA probe (10 ng/ml final concentration) per hybridization tube. For best results, use gel-purified DNA as probe. If scaling up probe preparation, increase number of tubes rather than increasing the amount of reagents per tube.
Probe preparation (0.5 mg DNA):
<ol>
<li>Dilute 15 &mu;l of cross-linker solution with 60 &mu;l of water.</li>
<li>Dilute DNA to 10 ng/&mu;l with water to 50 &mu;l total volume in a screw-capped microtube.</li>
<li>Denature DNA for 5 min in boiling water.</li>
<li>Rapidly transfer to ice and let cool for 5 min. Spin briefly.</li>
<li>Add 50 &mu;l of reaction buffer and mix gently.</li>
<li>Add 10 &mu;l of labeling reagent and mix gently.</li>
<li>Add 50 &mu;l of the diluted cross-linker solution from step 1 and mix gently. Vortex gently and spin briefly.</li>
<li>Incubate at 37&deg;C for 30 min.</li>
<li>Keep on ice for up to 2 h or add an equal volume of 100% glycerol and store at -20&deg;C. Note: Use the water supplied with the kit. Keep all reagents on ice unless otherwise stated.</li>
</ol>
Membrane preparation and hybridization
<ol>
<li>Preheat 50 ml of hybridization buffer and hybridization tube to 60&deg;C.</li>
<li>Add the hybridization buffer to the hybridization tube. Roll up membrane(s) and add to hybridization tube. (If adding 2 membranes per tube, first roll up one membrane completely, then roll up the other membrane on top in the same direction.)</li>
<li>Incubate hybridization tube at 60&deg;C for at least &frac12; h. (Make sure membrane(s) get soaked thoroughly.)</li>
<li>Transfer about 1 ml of hybridization buffer with a long pipette from the hybridization tube to the tube containing the labeled probe. Vortex gently and spin briefly. Transfer mixture back to hybridization tube.</li>
<li>Incubate at 60&deg;C overnight. Note: Use forceps and gloves when handling membranes. Mix content of hybridization tubes gently because too much agitation will cause permanent formation of undesirable foam. Membranes should be added in wet condition; soak in hybridization buffer or water if necessary and drain off excess liquid before adding.</li>
</ol>
2. Detection procedure
Washing membranes
<ol>
<li>Preheat primary washing buffer to 60&deg;C. (Use microwave oven and/or heating plate.)</li>
<li>If there are 2 membranes in one hybridization tube, transfer one membrane to a clean, preheated, empty hybridization tube.</li>
<li>Briefly rinse hybridization tube with about 50 ml of primary wash buffer.</li>
<li>Add about 100 ml of primary wash buffer to hybridization tube and incubate at 60&deg;C for 10&nbsp;min.</li>
<li>Repeat step 3 and 4.</li>
<li>Transfer membrane to washing container and incubate with at least 250 ml of secondary wash buffer at room temperature for 5 min.</li>
<li>Repeat step 6. Note: Membranes may be kept in the secondary wash buffer at room temperature for up to &frac12; h before signal generation. </li>
</ol>
Signal generation and detection (procedure for one membrane)
<ol>
<li>Tape two pieces of SaranWrap flat onto the tabletop (one for step 2 and one for step 4).</li>
<li>Carefully drain off excess buffer from the membrane by touching the corners of the membrane against the side of the container and transfer the membrane onto the SaranWrap (sample side up).</li>
<li>Pour 6-7 ml of ECF reagent over membrane and fold Saran wrap over membrane. Carefully distribute ECF reagent over membrane by gently moving a Kimwipe over the SaranWrap. Incubate for 5 min.</li>
<li>Drain off excess ECF reagent from the membrane and transfer it onto new SaranWrap, fold wrap over membrane, and seal the edges with tape. Cover the membrane with aluminum foil. Incubate for 1-2 hours.</li>
<li>Prepare the scanner glass plate by distributing 1-2 ml of ECF reagent over the glass plate. Then transfer the membrane onto the glass plate (sample side down) and distribute another 1-2 ml of ECF reagent over the membrane. Overlay with SaranWrap and carefully push all air bubbles away from the membrane. (You may place a stiff plate on top of the SaranWrap to keep membrane pressed against the glass plate.)</li>
<li>Use the Fuji FLA-5100 fluorescence scanner ("1 laser 1 image" mode) with the following settings: excitation 473 nm, detection filter LPB (510 nm), resolution 50 &mu;m, 16-bit signal graduation, CH1 400 V. (Scan takes about 15-20 min per membrane of size 22 by 22 cm.) Optional: </li>
<li>Let sit for 2-4 hours (or longer).</li>
<li>Repeat scan as in step 6.</li>
</ol>
3. Stripping and storing procedure (NUF version)
<ol>
<li>Transfer membrane to 100% ethanol and incubate at room temperature for 10 min with gentle agitation.</li>
<li>Replace with fresh 100% ethanol and incubate overnight at room temperature. (Membrane may remain in ethanol for several days.)</li>
<li>Replace ethanol with distilled water and incubate at room temperature for 10 min.</li>
<li>Add 100 ml water and 5 ml of 10% SDS (final 0.5% SDS) to a hybridization tube and mix gently. Add membrane.</li>
<li>Incubate at 80&deg;C for 1 h.</li>
<li>Transfer membrane to a clean, empty container.</li>
<li>Add 500 ml water and 5 ml of 10% SDS (final 0.1% SDS) a beaker and heat to a vigorous boil in the microwave oven.</li>
<li>Pour the near-boiling 0.1% SDS solution over the membrane and gently agitate for several minutes. Allow to cool to room temperature.</li>
<li>Rinse in at least 250 ml of sterile 100 mM Tris pH 8 for at least 10 min.</li>
<li>Wrap membrane in SaranWrap and carefully close edges with tapes. Store at 4&deg;C. (Membranes must never dry out.)</li>
</ol>

The stripping procedure has not been optimized. However, the stripping procedure given in the manufacturer's instruction (2 washes each, 10 min in 100% ethanol, 1 wash of 1 h at 60&deg;C in 0.5% SDS) is insufficient. At least overnight incubation in 100% ethanol is necessary to dissolve the ECF reaction product; several days incubation in 100% ethanol appears to have no adverse effects on the ability to reprobe the membrane. The manufacturer's SDS treatment also appears to be insufficient. NUF has tried 1 h at 80&deg;C w...

<table><tbody><tr><td>AlkPhos Direct Labelling Reagents</td><td>kit</td><td>Amersham</td><td>RPN3680</td><td>Contains labeling reagent, cross-linker solution, reaction buffer, water, control DNA, hybridization buffer, blocking reagent. Store hybridization buffer and blocking reagent at room temperature, other reagents at 2-8&amp;deg;C.</td></tr><tr><td>ECF Substrate</td><td>kit</td><td>Amersham</td><td>RPN3685</td><td>Contains ECF substrate, ECF substrate dilution buffer.Note: Pack contains total 60 ml reagent; aliquot into 10 ml samples in Falcon tube and store at 20&amp;deg;C. Use about 10 ml per membrane (size of 22 by 22 cm).</td></tr><tr><td>20x Secondary Wash Buffer</td><td>buffer</td><td></td><td></td><td>121 g Tris(final 1 M), 117 g NaCl(final 2 M).Adjust pH to 10. Adjust to 1 L with water. Store at 2-8&amp;deg;C for up to 4 months.</td></tr><tr><td>0.5 M NaH2PO4 buffer pH 7</td><td>buffer</td><td></td><td></td><td>69 g NaH2PO4.H2O. Adjust pH to 7 with NaOH. Adjust to 1 L with water. Store at room temperature.</td></tr><tr><td>1 M MgCl2</td><td></td><td></td><td></td><td>50.8 g MgCl2.6H2OAdjust to 250 ml with water. Store at room temperature.</td></tr><tr><td>10% (w/v) SDS</td><td></td><td></td><td></td><td>20 g SDSAdjust to 200 ml with water. Store at room temperatur</td></tr><tr><td colspan="5">Combination pack RPN3692 = RPN3680 + RPN3685</td></tr></tbody></table>

large-scale screens of metagenomic libraries

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

So our goal here is to inoculate colonies from some of these metagenomic libraries onto membrane filters, and each of these filters can hold 10, 000 colonies. So what rums doing right now is laying this nylon filter onto a block and kind of smoothing it out. It's, it's in a little bit of liquid, it's just the, the growth media luria broth.And what he's then going to do is transfer this moistened membrane over to the robot that is gonna automatically inoculate this membrane with 10, 000 colonies. And robotics are essential for this kind of procedure because no human being really has the patients or accuracy to spot 10, 000 colonies onto one of these membranes. Robots are one of the things that has actually advanced genome science by allowing very repetitious and, and kind of small spatial scale operations to be done with high accuracy.Right now, rum setting up the robot to feed these 3 84 well plates each well in those 3 84 well plates holds an individual colony from the Metagenomic library. Right now, the robots sterilizing the inoculating needles that it's going to use to spot on the membrane. So it goes through several different solutions, one of which is 70%ethanol, kind of sterilize it, and then those inoculating needles are gonna get dipped into a 3 84 well plate that has all the colonies.Now it's being sterilized and dried by light. After the inoculating needles are dried, a plate will get fed through the robot. Each of those colonies is actually blotted and duplicate, which helps as a control.So we're actually looking for two colonies that are the identical ones to show a signal from the hybridization. After all those colonies have been spotted on the filter. Again, it's applied to the augurous plate that you see here, and then they just put at 37 degrees C overnight.What happens is the colonies each grow up and what you see there array are 10, 000 colonies on a filter. Now of course, each one of those colonies has many, many, many copies of the recombinant DNA, the metagenomic DNA in this case. This is from the central North Pacific Ocean that we made.What Saltram will do next is transfer that membrane into a series of solutions. The first solution he transfers to is a basic solution of sodium hydroxide, and that's gonna lyce the colonies. It basically dissolves the cell wall and frees the DNA so that it can, the DNA makes contact with the filter.And so what's happening here is all the cell walls of all the bacteria in those colonies are dissolving and the DNA is now free. After five minutes in sodium hydroxide, the filters then transferred. This is just a place to rest it because next it's gonna go in boiling steam.So this is the second part of the lysis step. First, the cells have been treated with a highly basic solution of sodium hydroxide and detergent sodium doude sulfate. Now those colonies are being subjected to high heat, steam heat a hundred degrees Celsius.The combination of that base and detergent and this heat will totally ly the cells and the DNA will be free upon the filter. After five minutes in the steam ER's, neck's gonna transfer that filter to a neutralizing solution. The neutralizing solution basically neutralizes all that base and then the filter will be dried.So now this is the neutralizing solution, and that sits in the neutralizing solution for another five minutes. The filter's next transferred to blotting paper and allowed to dry typically overnight. After the membranes with the lys cells have been dried overnight, they're placed in a solution of triss, EDTA and protease K, and the protease K is to remove all the protein that's that's on the membrane.You see rum has flipped it over to make sure that all those colonies are getting coated with the protease K.That's gonna digest a lot of the extraneous cellular material and the protein. And after a half an hour's incubation in protease K at 37 degrees C, the membranes are just taken out of solution and dried on blotting paper overnight. And then they're ready for the next step, which is the hybridization.My name is Vin fm. I'm a postdoc in at delong's Lab. At MITI use macro array technology to screen for phylogenetic markers and also functional genes.You've been shown how those macro array array libraries were prepared. I'm going to walk you through the rest of the procedure on how to do the screening. Here's the a macro array we, before we can proceed, we need to fix the DNA onto the membrane itself by cross-linking with uv.We do that with a UV strata linker, put that in there, set that for autocross link and start. So once it's been cross-linked, what I like to do is cut the membranes into two halves. I do that because these membranes will be hybridized inside these hybridization tubes and cutting them in half allows the entire inside surface of the membrane to be fully exposed to the hybridization solution.You can see that the membrane sits perfectly inside the tube without rolling over on itself. So here we've got the membrane nicely housed inside the hybridization tube. Next thing is I'm gonna add hybridization buffer to it.Containing probe, which I've got in this other tube that was preheated at 60 degrees. That's my preferred hybridization temperature. So before I do that, well, while I do that, I'll explain that the hybridization buffer sent to us with our detection kit by manufacturer.It says hybridization buffer, which we supplement with blocking reagent. Also included is phosphate buffer, SDS, magnesium chloride and sodium chloride, plus some urea.Great. So the next thing is putting this tube with its hybridization buffer membrane and probe inside the hybridization oven, which I've set up to be 60 degrees and set it and forget it for an overnight incubation.So after ideally an overnight incubation, it's time to wash the membranes to remove any unbound probe. There are no hazardous materials in the preparation of the hybridization buffer or the probe. So it's safe to dump it down the sink.I'm gonna go grab my primary wash buffer, which is warming up at 60 degrees as well. It's been heating up nicely at the same temperature at which the membranes have been hybridizing overnight. I, it's going to be washed at the same temperature as well.So you, I usually do three washes with the primary wash buffer with each incubation of five to 10 minutes roughly. And again, the wash buffer is non-radioactive known hazardous chemicals after each wash cycle, you just dump, dump that down the sink as well. It's been three washes when primary wash buffer at 60 degrees.Now the membranes are ready for the secondary wash, which brings the membranes to the proper pH for exposure to the ECF detection chemicals. So next we wash membranes in secondary wash buffer at room temperature. Now you can use tweezers to remove the membranes or if you're very careful, you can use your gloved hand and handle the membranes only either on the back side or at the edges so that you don't smudge the membranes with any contaminants that might be on your, your hands or even when they're gloved.We should transfer the membranes into a container that contains a secondary wash buffer preport and been brought to room temperature. Just lay it down nice and flat. Sometimes the membranes will overlap, but it's not that important.Remember this particular step just equilibrates the pH for the membranes. Then you let that incubate. You can do a little rocking if you want to.I have not found that to be particularly useful or harmful either way. So I just leave it on my bench for five to 10 minutes again, and then I repeat the secondary wash for a second time. Second wash of the secondary wash is done.Now it's time to set up for detection, expand. So I'm threading out some saran wrap and the membranes will be laid on the saran wrap with the detection reagents port on top of the membranes and then fold the saran wrap back over. And that creates a nice little pouch to hold the detection reagent.Here we've got our membranes. I drain any excess wash buffer onto some dry paper towels, a stack of them, and lay the membranes out onto Theran wrap. You have got some ECF detection pre-mixed.These can usually be stored in the freezer for several months on end, I just pour the reagents onto the membrane. I I fold the leftover. So ran wrap over.And I create a little bit of a, a pouch by tucking in the ends or the edges and protect the whole detection process with some aluminum foil. And I leave that for about five to 10 minutes as well. It's time to drain the detection chemicals from this first bag.You notice that I, I taped part of the clinging wrap onto my bench. You, you might find that to be useful oftentimes to prevent the, the kind of sticking that cera wrap is bound to do. Again, it's helpful to drain by dapping onto a stack of paper towels unroll into the fresh, clean, clean wrap that you've pre set onto the bench.Okay, so detection chemicals are gone for the most part for, okay, so membranes are ready for about a two hour incubation to let the signal develop. So it's been two hours and we're ready to detect the signal on our membranes. And I, we use that by scanning the membranes using a fluorescent scanner, again, only handling the membranes at the edges.What you might find useful is to use a pipette to roll out any bubbles that form between the membrane and the glass. So we're ready to do the scanning. Set up the area that was covered on the face plate by our membranes and scan at four and 73 nanometers.So when you blow up a finished scan, you'll get something similar to this image. You've got a signal right there. Whenever you have two opposing dots, that's the sign of a positive signal.Here's one example, here's a second example. So once you know what part of the membrane has the signal, you then retrace which plate and which wells the particular signal corresponds to. That will tell you which fo smidt is carrying the particular gene that you were trying to identify with your screen.And once you identify the smid, you can look for you. One way is just to sequence the entire smid is insert and find that gene that you're interested in.

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Learn robotic inoculation of metagenomic libraries onto membrane filters for high-throughput genomics. This method enables accurate placement of 10,000 colonies per filter, advancing microbial community research.

Large-Scale Screens of Metagenomic Libraries

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