TITLE: No Title
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[DESCRIPTION]
As used in Rich, Konstantinidis, and DeLong. 2008.&nbsp;<br /><br />Rich VI., Konstantinidis K., DeLong EF.&nbsp;Design and testing of 'genome-proxy' microarrays to profile marine microbial communities. <a href="http://www.ncbi.nlm.nih.gov/pubmed/18028413" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed/18028413</a>.&nbsp;Environ. Microbiol., 2008 Feb;10(2):506-21. Epub 2007 Nov 19.&nbsp;<br /><br />

[GUIDELINES]
<strong>Materials<br /></strong>-2 full ice buckets<br />-0.2 &micro;m syringe-filters (e.g. Pall Corp. #4192, Acrodisc 25 mm syringe filters, sterile)<br />-syringes, size depends on volume buffer required; usually 5 ml, 20 ml and 60 ml<br />-rotating hyb oven set to 37&deg;C<br />-rotating hyb oven set to 55&deg;C<br />-later, a heat block or hyb oven (depending on your lysis set up) set to 70&deg;C<br />-96 well DNA size exclusion columns, (e.g. Edge Biosystems ExcelaPure 96 well UF Plate)<br />-Qiagen DNeasy 96 Tissue Kit, Qiagen #69581<br />-1 M Tris, pH 8.3 (kept at my bench, but pH 8.0 - this is OK; e.g. Ambion #9855G)<br />-0.5 M EDTA (kept at my bench, e.g. Ambion #9260G)<br />-Sucrose (e.g. stockroom; Mallinckrodt Chemicals #8360-04)<br />-10% SDS (kept at my bench; e.g. Ambion #9822)<br />-Lysozyme (kept in door of freezer; e.g. Sigma #L-6876)<br />-Proteinase K (stored frozen and dry; e.g. EMD #24568-2)<br />-100 mg/ml RNase (stored at RT, in bench drawer; e.g. Qiagen #1018048)<br /><strong><br />Protocols relevant to the development of this one:</strong><br /><br />-The lab's latest 'DNA/RNA Extraction from Large Volume Steripak Filters' protocol, used after the recent Hawaii cruise to extract community DNA for library making.&nbsp;<br /><br />-The extraction protocol described in Suzuki et al. 2001, the upwelling plume paper; they extracted DNA from 13 mm Supor-200 filters used to filter 30 ml of seawater. They froze filters in 180 &micro;l of a homemade lysis buffer: 20 mM Tris, pH 8; 2 mM EDTA, pH 8, 1.2% TritonX, and 20 mg/ml lysozyme. Once thawed at a later time, they incubated 1 hr at 37&deg;C. Added 1 &micro;l of 15 Kunitz U/ml RNase A, incubated 5' @ RT. Then added 25 &micro;l of a 25 mg/ml ProK (final conc. 3.05 mg/ml). Mixed by vortexing, then followed Qiagen DNeasy Tissue Kit, protocol for Gram-positive bacteria: incubate 1-3 hrs at 55&deg;C, vortex 15 sec, add 200 &micro;l BufferAl (this contains guanidine salt and Tween), incubate 70&deg;C 10', add 200 &micro;l 96-100% EtOH, mix thoroughly by vortexing, and apply to columns.<br /><br />-Bostr&ouml;m et al. 2004 (Ake Hagstr&ouml;m's Lab) L&amp;O Methods paper, vol. 2:365-373. Optimization of DNA extraction for quantitative marine bacterioplankton community analysis. Their lysis buffer was 400 mM NaCl, 750 mM sucrose, 20 mM EDTA, 50 mMTris pH 9, and 1 mg/ml final conc. lysozyme. Incubated 30' at 37&deg;C (they also tried 120' but saw worse extraction efficiency, prob due to nuclease activity Tracy thought). Then added SDS to final conc. of 1%, and ProK to final conc. of 100 &micro;g/ml. They incubated at 55&deg;C and saw that an overnight treatment was far better than a shorter treatment. They then proceed with a phenol extraction protocol. Didn't end up using their protocol but mostly because the Steripak one wasn't too diff, and although it only has a 2 hr ProK incubation, it also has a 6.5x higher ProK conc.<br /><br />-Conversations with Tracy. He typically used 1 mg/ml final lysozyme conc., 200 &micro;g/ml RNase.<br /><br />- DNeasy 96 Tissue Handbook protocol.<br /><br />-ExcelaPure 96-well UF PCR Purification protocol.<br /><br /><strong>Expected DNA yield:</strong>&nbsp;<br /><br />Rule of thumb is about 1 fg per genome, 10<sup>6</sup> genomes per ml, so 1 ng DNA per ml, so 1 &micro;g DNA per L. SO, I filtered 250 mls - 1 L, so I shoud expect (best case scenario) 250 ng - 1 &micro;g DNA out. <br /><br /><strong>Sources by step:</strong><br /><br />Cell Lysis &amp; RNA Removal: adapted from lab Steripak protocol<br />Protein Degradation: adapted from lab Steripak protocol <br />DNA Purification Through DNA-binding Columns: adapted from DNeasy Tissue kit protocol for Gram-positive bacteria, and from DNeasy 96 Tissue kit protocol, and from Marcelino's protocol in the upwelling paper<br />Final DNA Clean-up &amp; Concentration by Size-Exclusion Columns: from the ExcelaPure 96-well UF PCR purification kit protocol<br /><br /><strong>Purification Through DNA binding Columns Notes:</strong><br /><br />Principle: selective adsorption of DNA to a silica gel membrane<br />Notes: According to 96 kit, vol. after steps 1&amp;2 would be about 200 &micro;l, to which they add 410 of buffer AL/E; according to Marcelino's protocol with the old Tissue kit, after steps 1&amp;2 vol. would be 206 &micro;l, to which they added 200 &micro;l buffer AL, incubated 10' at 70, then added 200 &micro;l EtOH. Tech support for 96 kit says to use the slightly higher ratio of buffer to lysate vol that they use in their protocol. They also confirmed that Buffer AL/E, before you add the ethanol, is the same as buffer AL from the old kit.<br /><br />Note on spins: these are very fast spins, and so balancing is important. If only doing 1 plate, Tracy suggests using a 96 deep well plate as a balance (water squirt bottle). He says it will shake anyway but do not be alarmed. He greased the hinges of the swinging plate holders and said they should be good until next year (2008), and that the plates do fit, barely, in our rig with room to rotate. &nbsp;<br /><br />The max speed of our rotor is hypothetically 5650 rpm but it will only accept 5250 rpm; 5000 rpm= '4612xg' on the display. So, while the 96 well protocol calls for faster spins, we can't reach them.&nbsp;<br /><br />Our centrifuge in the main lab does not really recognize the plate spinning rotor; therefore, it does not correctly convert the rpm to the rcf. You can trust the rpm, but not the rcf; that you should calculate yourself.&nbsp;<br /><br />The equation for interconverting RCF (xg) and RPM is:<br />RCF=(1.12*radius in mm)*(RPM/1000)<sup>2</sup><br /><br />Thus,Mix our 5000 rpm spins on our rotor, which has a max plate spinning radius of 16 cm but which spins the DNeasy plates at ~12 cm, provides 3360 xg.<br /><br /><strong>Final DNA Clean up &amp; Concentration by Size Exclusion Columns Notes:&nbsp;</strong><br /><br />Excelapure protocol calls for 100 &micro;l elution, Tracy has gone as low as 20 &micro;l.<br /><br />Dilute TE is 1:10 regular TE.<br /><br />Expected DNA yield: for a 250 ml filtration, if only on the order of 10<sup>5</sup> cells (as in most Hawaii samples, or for sparse MB samples) then only 25 ng of DNA, so 1.25 ng/&micro;l. DNA stores better at higher conc. too.&nbsp;<br /><br />&nbsp;<br /><br /><br /><br /><br /><br />

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