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P1 bead enrichment
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NB! post-PCR operation.
This protocol is for beads, collected from ONE plate / TWO platesTWO plates / ONE plate
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Equipment and consumables
- vortex;
- 45°-cut 200µl tips;
- magnetic stand;
- powder-free gloves;
- LoBind tubes & tips;
- rotator at NT;
- 61°C water bath;
- (optional) Covaris S2:
- change water;
- start degassing;
- prepare post-PCR tube holder;
- (i) melt, (ii) vortex, (iii) spin-down and (iv) put in ice-box 1mM Enrichment Oligo (should not be diluted) 3,5µl per plate;
- prepare 1.7ml tubes, for each library:
- 4x with 300µl 6x with 400µl with 60% Glyc., labelled #lib;
- 26x with 1ml of TEX, labelled #lib;
- prepare buffers (if process more than 4 libraries, otherwise do it during EB-charging):
◊ 60% Glyc. (60% glycerol, fresh weekly): vortex to mix well, store in ice;
◊ DenBuffer (fresh weekly): mix well, store at NT;
- prepare 1.7ml tubes, for each library:
- 4x with 300µl 6x with 400µl with 60% Glyc., labelled #lib;
- 26x with 1ml of TEX, labelled #lib
E-beads charging
- E-beads aliquot:
- vortex E-beads (30 sec);
- put 650µl1.3ml of E-beads into 1.7ml tube #EB_lib;
- centrifuge 1 min at 14,000xg & remove supernatant;
- two times Bi-buf washing:
◊ ◊ re-suspend E-beads in 900µl1.3ml of Bi-buf: vortex 20 sec;
◊ ◊ centrifuge 1 min at 14,000xg & remove supernatant;
- charging by Enrichment Oligo:
- re-suspend E-beads in 350700µl of Bi-buf: vortex 20 sec & pulse-spin;
- add 3.57µl of 1mM Enrichment Oligo;
- vortex E-beads 30 sec;
- rotate E-beads at NT for 30 min:
start-time: _________
finish-time: _________
- TEX washing (pour out the first portion):
- centrifuge 1 min at 14,000xg & remove supernatant;
- two times TEX washing:
◊ ◊ re-suspend E-beads in 900µl1.3ml of TEX: vortex 20 sec;
◊ ◊ centrifuge 1 min at 14,000xg & remove supernatant;
- LS: transfer E-beads in 150300µl of LS: vortex 30 sec & pulse-spin
Preparation of single-stranded P1 beads
- concentrate:
- pulse-spin;
- magnetic rack (1 min) & remove supernatant;
- two times denaturation:
◊ ◊ re-suspend the beads in 300600µl DenBuffer: vortex 20 sec;
◊ ◊ incubate 1 min & pulse-spin;
◊ ◊ magnetic rack 1 min & remove supernatant;
- two times washing:
◊ ◊ re-suspend the beads in 300600µl of TEX: vortex 20 sec & pulse-spin;
◊ ◊ magnetic rack (1 min) & remove supernatant;
Hybridization
NB! NO ANY MAGNETS up to §15
- re-suspend the beads in 150300µl of TEX:
- vortex 20 sec & pulse-spin;
- Covaris S2: 2x "Covalent Declump 1"/2;
- pulse-spin;
- combine P1- and E-beads:
- add E-beads to P1-beads in correspondent #lib tubes;
- vortex (30 sec) & pulse-spin;
- Covaris S2: "Covalent Declump 3";
- shake down (do not spin!);
- hybridization of P1- and E-beads:
- incubate at 61°C for 15 min, vortexing (20 sec) and shake down every 5 min:
time 1: _______ _______ _______ _______ _______ _______
time 2: _______ _______ _______ _______ _______ _______
time 3: _______ _______ _______ _______ _______ _______
- cool beads in liquid ice (2 min);
- enrichment:
- vortex (10 sec) & pulse-spin;
- mix by pipetting & ACCURATELY load 75100µl on top of the 60% glycerol in 1.7ml #lib tubes;
- centrifuge 3 min at 14,000xg;
- using one tip per one library collect TOP layers from the hinged sides of #lib tubes and put them into BOTTOMS of twosix #lib tubes (upper solution is for the rinsing of tip);
- TEX washing:
- vortex #lib tubes (20 sec);
- centrifuge 1 min at 14,000xg & remove supernatant;
- re-suspend the beads in 400300µl of TEX: vortex 20 sec;
- collect in two 1.7ml tubes;
- centrifuge 1 min at 14,000xg & remove supernatant;
- re-suspend by pipetting the beads in 100µl of DenBuffer, (ii) collect content of two #lib tubes in one, (iii) use additional 100µl DenBuffer to wash tubes and tip;
NB! MAGNETIC RACK starts here
Washing
- denaturation:
--- repeat until supernatant is absolutely clean (normally three times; only tiny white blur should be in solution during the second washing, third washing should be practically clean) ---
◊ ◊ ◊ pulse-spin & magnetic rack (1 min);
◊ ◊ ◊ ACCURATELY remove supernatant with E-beads;
◊ ◊ ◊ add 400800µl of DenBuffer & vortex 20 sec;
◊ ◊ ◊ incubate 1 min;
- two times TEX-washing:
◊ ◊ re-suspend the beads in 400800µl of TEX: vortex 20 sec & pulse-spin;
◊ ◊ magnetic rack 1 min & remove supernatant;
- declumping:
- resuspend the beads in 400800µl of TEX: vortex 1 min & pulse-spin;
- Covaris S2: "Covalent Declump 1";
- pulse-spin;
- secondary TEX-washing:
--- repeat until supernatant is absolutely clean (at least two times) ---
◊ ◊ magnetic rack 1 min & remove supernatant;
◊ ◊ re-suspend the beads in 400800µl of TEX: vortex 20 sec & pulse-spin;
Notes
- beads should have ~95% purity after enrichment;
- charged Enrichment Beads should be used within one week (store in TEX at 4°C);
- removal of supernatant from uncharged Enrichment beads should be done in three steps:
- centrifuge 1 min at 14,000xg;
- turn tubes in the rotor on 180° to put pellet down;
- wait 1.5 min until beads went down;
- remove ~80% of supernatant (until beads come to the tip);
- slightly shake tube to wash beads from the wall;
- repeat above procedure twice with shorter centrifugation: 0.5 min at 14,000xg;
§§ 9-14
- it is convenient to process two-tubes portions.
Second-generation sequencing
URL: http://seq.zbio.net
e-mail: soldatov@molgen.mpg.de
visits:
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Second-generation sequencing > Bead enrichment
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