TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="font-weight: 400;">Most noncommercial tradition DNA extraction protocols result in a crude DNA preparation. If the DNA is intended to be used for a high-end application like Nanopore sequencing, it requires a thorough clean-up and size selection before it could be used for sequencing. </span></p>
<p><span style="font-weight: 400;">Solid Phase Reversible Immobilisation (SPRI) magnetic beads is a quick and convenient way of purifying and size selecting intact double-stranded DNA from crude DNA. Most commercially available SPRI beads based DNA purification mix is quite expensive so our lab endeavored to develop an inexpensive beads mix which is as good as the commercially available ones. In this effort, our lab has optimized a beads mix for purifying and size selecting crude DNA extracted from eucalyptus and posted on protocol.io, https://www.protocols.io/edit/high-purity-high-molecular-weight-dna-extraction-f-n5ydg7w?step=16.</span></p>
<p><span style="font-weight: 400;">However, this solution was not very effective in purifying crude DNA extracted from fungal material. DNA extracted from fungal material is highly viscous which is indicative of high levels of impurities in the DNA preparation. I tried to improve the beads mix for purifying rust DNA by adding 0.25 % (v/v) Tween-20 into the beads mix.</span></p>
<p><span style="font-weight: 400;">I tested beads mix with Tween-20 to see if adding tween into the solution makes any difference in the recovery, purity and size selection.  Turns out that bead solutions with Tween-20 make big difference in the size selection and recovery of the DNA compared to the bead solutions without.  </span></p>
<p> </p>
<p><span style="font-weight: 400;">I calibrated/tested the bead solution with and without Tween-20 on the 1 kb DNA ladder to establish which DNA solution to beads volume ratio gives optimal recovery and size selection. We found that beads mix with 0.25 % Tween-20 works much better in size selection and recovery than beads mix without Tween-20. The best DNA to beads volume ratios were  1.0: 0.9 and 1.0: 1.0. </span></p>
<p> </p>

[GUIDELINES]
<p> </p>
<p>Beads protocol was originally adapted from Philippe Jolivet and Joseph W. Foley, 2015 - Solutions for purifying nucleic acids by solid-phase reversible immobilization (SPRI)</p>
<p>Link: http://www.openwetware.org/images/f/f8/SPRI_buffers_v2_2.pdf </p>
<p> </p>
<p><span style="font-weight: 400;">Calibrating the newly prepared beads mix: Every time you prepare new a fresh beads mix, make sure you test it with either ladder DNA or DNA which you don't mind losing.</span></p>
<p> </p>
<p><span style="font-weight: 400;">pH makes a huge difference in the solubility of DNA and beads. If not set properly, beads tend to clump at DNA elution step that could lose as much 60-70 percent of the DNA. So make sure that your elution buffer (e.g. 10 mM Tris or 0.1 x TE) has a pH of 8.  </span></p>
<p> </p>
<p><span style="font-weight: 400;">Homogeneous beads mix at room temperature before use.</span></p>
<p> </p>
<p><span style="font-weight: 400;">Always use freshly prepared  70 % Ethanol </span></p>
<p><span style="font-weight: 400;"> Preheat your elution buffer of choice (TE-Buffer, Tris 10 mM, Water..) to 37-42°.</span></p>
<p> </p>
<p>We use same beads solution for the MinION sequencing library preparation. </p>

[STEPS]
SECTION: Make beads stock solution
?. 
SECTION: Make beads stock solution
?. 
SECTION: Make beads stock solution
?. 
SECTION: Make beads stock solution
?. 
SECTION: Make beads stock solution
?. 
SECTION: Make beads stock solution
?. 
SECTION: Make beads stock solution
?. 
SECTION: Make beads stock solution
?. 
SECTION: Clean up
?. 
SECTION: Clean up
?. 
SECTION: Clean up
?. 
SECTION: Clean up
?. 
SECTION: Clean up
?. 
SECTION: Clean up
?. 
SECTION: Clean up
?. 
SECTION: Clean up
?. 
SECTION: Clean up
?. 
SECTION: Clean up
?. 
SECTION: Clean up
?. 
SECTION: Clean up
?. 
SECTION: Clean up
?. 
SECTION: Clean up
?. 
SECTION: Clean up
?. 
SECTION: Quality control
?. 
SECTION: Quality control
?. 
SECTION: Make beads stock solution
?.