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Blunt end DNased DNA in-gel(For low cook gel alertness of DNase advised DNA, see Crawford et al., Genome Research 2006)

Plastic Tube Labeling Machine,Labeler - YouTube - eppendorf tube labeling machine
Plastic Tube Labeling Machine,Labeler – YouTube – eppendorf tube labeling machine | eppendorf tube labeling machine

1) Ablution abundantly in T4 DNA Polymerase Absorber ( DTT) , 3×50mls for 1 hour anniversary (to get rid of EDTA in plug)2) Aish all aqueous from 50 ml conical tube and add Polymerase mix: 80µl DNA bung (low cook gel); 12µl 10x Polymerase Absorber (NEB#2); 5 µl dNTPs (10uM; NEB Cat # M0203L); 6 µl T4 DNA Polymerase ; 99.2 µl H20; 2 µl BSA (100x)3) Incubate at allowance acting for 3-4 hours (gently mix every hour or so)4) Add bung to 500 μl TE5) Calefaction to 65 oC for 10 account (flick every brace of account to deliquesce agarose)

Phenol: Phenol/Chloroform: Chloroform abstract (using wide-bore tips)

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6) Add 1/10 aggregate 3M NaOAc, 2 volumes ETOH, and 1µl glycogen (Roche Cat# 901393)7) Place in freezer for at atomic 30 minutes8) Circuit 15 account at 4 deg and ablution 1x with 70% ETOH9) Remove, quick spin, and aish of liquid10) Air dry for 5 account (NO LONGER!)11) Resuspend in 40 μl TE

Ligation of biotinylated linkers12) Incubate 2µg DNA, 10µl 5x Ligase Absorber (use advanced apathetic tips; Invitrogen cat # 46300-018), 6.7µl Annealed linkers (102B/103A; THIS IS OLIGO SET A… THAW LINKERS ON ICE), 0.5µl T4 ligase (NED Cat # M0202L), H2O to 50 µl at 16 oC overnight.13) Shear DNA by abacus articulation to 1.5 mls TE (in 15 ml conical), putting on ice and sonicating on ambience 3 for 25 additional pulses (sonicate while tube is in ice bath)14) Pulse anniversary sample 8 times (back on ice afterwards anniversary sonication). The tip should be about at the basal of tube, with no movement. Cool the sonicator tip afterwards anniversary annular with an ice bath

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DYMO Compatible Laboratory Labels – YouTube – eppendorf tube labeling machine | eppendorf tube labeling machine

Bind to Streptavidin beads15) Use 100 ul of chaplet per reaction16) FIRST Ablution chaplet 3×1ml in binding/wash absorber (TE additional 1M NaCl)17) Add 300 μl 5M NaCl to sonicated DNA (final absorption of 1M NaCl)18) Add 100 μl of chaplet to sonicated DNA19) Rock for 15 account at allowance temp20) Use allurement to abduction biotinylated ends21) Ablution 3×1ml in binding/wash absorber (TE additional 1M NaCl)22) Resuspend chaplet in edgeless catastrophe mix (see aing section)

Blunt end sheared ends23) Incubate 97.3 μl H2O, 11 μl T4 Absorber (NEB absorber 2), 1 μl 10mM dNTPs (Roche # 11 814 362 001), 0.2 μl T4 DNA Polymerase (NEB Cat # M0203L) and 0.5 μl 100x BSA for 1 hour at 16 oC (resuspend chaplet already during incubation)24) Ablution chaplet 3×1ml in binding/wash absorber (TE additional 1M NaCl)25) Resuspend chaplet in 50 μl articulation mix

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tube labeling | tube printing machine | lab label printing .. | eppendorf tube labeling machine

Ligation of non-biotinylated linkers to beads26) Mix 32.8 μl H20, 10 μl 5x Ligase Absorber (use advanced bore tips; Invitrogen cat # 46300-018), 6.7 μl Annealed linkers (102C/103B; THIS IS OLIGO SET C…THAW LINKERS ON ICE) and 0.5 μl T4 Ligase (NEB Cat # M0202L).27) Add mix anon to chaplet and resuspend28) Incubate at 16 degrees O/N (resuspend chaplet already during incubation)

Clean Streptavidin beads29) Ablution 3×1 ml in binding/wash absorber (TE additional 1M NaCl)30) Resuspend pellet in 50 μl TE pH 8.0

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LM-PCR Reaction31) Mix 32.5 μl H20, 4 μl 10x ThermoPol Buffer, 1.25 μl dNTPs (10uM; Roche # 11 814 362 001), 1.25 μl oligo OJW102 (40μM, 102C) and 1 μl beads.32) Place in a PCR apparatus and calefaction up the PCR apparatus to 50 deg, put on authority and add the following: 0.5 μl Taq DNA Polymerase 0.5 (Invitrogen cat# 18038-042), 8.5μl H20, and 1μl ThermoPol Absorber 1 (NEB Cat #B90045)

33) Cycle application the afterward conditions:55×4 min (add Taq at this date – hot start)72×3 min95×2 min25 X (95×30 sec, 60×30 sec, 72×1 min)72×5 min4 x forever

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Clean up of LM-PCR product34) Add 400 μl TE to pcr product35) Do a phenol/chloroform abstraction application appearance locks 2.0 ml (Brinkmann Instruments. Description: Eppendorf appearance lock 2 ml Cat # 62111-400)36) ETOH ppt with 1 μl glycogen (Roche Cat# 901393)37) Resuspend pellet in 25 μl TE38) Clean up on ArrayIT columns microarray delving ablution kit (Cat #FPP $70 for 50 columns)39) Elute with 50 μl water

Quantitate, label, and blend to arrays

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Automated Tube Labeling and Microplate Labeling – Scinomix – eppendorf tube labeling machine | eppendorf tube labeling machine

Use the afterward Oligos:1) oJW102A: 5’ biotin TEG- GCG GTG ACC CGG GAG ATC TGA ATT C –3’2) oJW102B: 5’ /5Bio/ GCG GTG ACC CGG GAG ATC TGA ATT C –3’3) oJW102C: 5’ GCG GTG ACC CGG GAG ATC TGA ATT C –3’4) oJW103A: 5’ /5Phos/GAA TTC AGA TC/3AmM/ -3’5) oJW103B: 5’ GAA TTC AGA TC -3’

Oligo SET A: 102B/103A Oligo SET C: 102C/103B

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Centrifuge Test Tube Labeling – TubeWriter11 – eppendorf tube labeling machine | eppendorf tube labeling machine

40) Anneal primers by bond 50 μl Tris-HCL 1M, pH 7.6; 375 μl of 40 μM oJW102; 375 μl of 40 μM oJW103 and 200 μl H2O and putting the mix in baking water, shutting off heat, and absolution oligos sit in baptize until allowance acting is reached.41) Put at 4 degrees O/N. Aliquot and abundance at -20. To anticipate primers from acceptable un-annealed, consistently thaw annealed primers on ice.

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Vial Labels | Vial Labeling | Vial Labeler – Tubewriter | eppendorf tube labeling machine
Vial Labels | Vial Labeling | Vial Labeler - Tubewriter
Vial Labels | Vial Labeling | Vial Labeler – Tubewriter | eppendorf tube labeling machine

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