AlanFung:LabNotes/Probe/2013-2-1

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[1]

Expansion PCR[edit]

  • We will set up 8 reactions for each primer-oligo set (8 in total)
  per reaction V4 Set1 V4 Set2 V4 Set3 V4 Set4
20nM Oligo Pool 0.2 1.6 1.6 1.6 1.6
2X Kapa SYBR qPCR MM 50 400 400 400 400
100uM pAP1V41U 0.4 3.2 3.2 3.2 3.2
100uM AP2V4 0.4 3.2 3.2 3.2 3.2
2mM aa-dUTP 3 24 24 24 24
H2O 46 368 368 368 368
Total Volume 100 800 800 800 800
           
  per reaction V6 Set1 V6 Set2 V6 Set3 V6 Set4
20nM Oligo Pool 0.2 1.6 1.6 1.6 1.6
2X Kapa SYBR qPCR MM 50 400 400 400 400
100uM pAP1V61U 0.4 3.2 3.2 3.2 3.2
100uM AP2V6 0.4 3.2 3.2 3.2 3.2
2mM aa-dUTP 3 24 24 24 24
H2O 46 368 368 368 368
Total Volume 100 800 800 800 800
95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 16 -> 72C 2min -> 15C hold.

QPCR Results[edit]

EtOH Precipitation[edit]

  • Pool all reactions into 15ml tube (900ul in total)
  • add 3ul of glycoblue
  • add 90ul of 3M NaoAc
  • add 2250ul of 100% EtOH
  • Freeze the tube in -80C for 1hr
  • centrifuge at 3000rpm for 30min, 4C
  • remove supernatant and add 600ul of chilled 75% ETOH and transfer the DNA pellet to a 1.5ml tube (5000ul-3750ul100% EtOH + 1250ul ddh2o)
  • spin at 14,000rpm for 5min
  • dry the pellet in the hood and resuspend w 100ul ddh2o
  • re-purify the amplicons with 2 Qiaquick columns, elute w 40ul EB for each column
  • measure concentration w nanodrop

Nanodrop Results[edit]

Probe Set ng/ul ul ng
V4S1 27 80 2160
V4S2 47.7 80 3816
V4S3 61.6 80 4928
V4S4 35.9 80 2872
V6S1 61.2 80 4896
V6S2 49.7 80 3976
V6S3 74.9 80 5992
V6S4 86.7 80 6936

DpnII Digestion[edit]

  • We will setup a small testing reaction to optimize the restriction endonuclease reaction
  • I will test the reaction with 1ug of probes
Probe Set ng/ul To get 1ug 10X DpnII Buffer DpnII H2O Total
V4S1 27 37.04 5 1 6.96 50
V4S2 47.7 20.96 5 1 23.04 50
V4S3 61.6 16.23 5 1 27.77 50
V4S4 35.9 27.86 5 1 16.14 50
V6S1 61.2 16.34 5 1 27.66 50
V6S2 49.7 20.12 5 1 23.88 50
V6S3 74.9 13.35 5 1 30.65 50
V6S4 86.7 11.53 5 1 32.47 50
  • Incubate reaction at 37C for 1 hour followed by 20min at 65C for heat inactivation
  • Column purification, elute with 30ul EB

PAGE Gel[edit]

File:ZhangLab 2 2013-02-04 14hr 19min.jpg

DpnII Digestion for remaining probes[edit]

Probe Set ng/ul Remaining Volume 10X DpnII Buffer DpnII H2O Total
V4S1 27 42.96 10 5 42.04 100
V4S2 47.7 59.04 10 6 24.96 100
V4S3 61.6 63.77 10 7 19.23 100
V4S4 35.9 52.14 10 6 31.86 100
V6S1 61.2 63.66 10 7 19.34 100
V6S2 49.7 59.88 10 6 24.12 100
V6S3 74.9 66.65 10 7 16.35 100
V6S4 86.7 68.47 10 7 14.53 100
  • Incubate reaction at 37C for 1 hour followed by 20min at 65C for heat inactivation
  • Column purification, elute with 30ul EB
  • combine the two sets of probes, making the total volume 60ul
Probe Set Concentration (ng/ul)
V4S1 20.5
V4S2 40.7
V4S3 57.7
V4S4 32.5
V6S1 53
V6S2 45
V6S3 71.2
V6S4 83.1

Lambda exonuclease digestion[edit]

Probe Set Volume 10X Lambda Exo Buffer Lambda Exo (10U/ul) H20
V4S1 60 10 5 25
V4S2 60 10 5 25
V4S3 60 10 5 25
V4S4 60 10 5 25
V6S1 60 10 5 25
V6S2 60 10 5 25
V6S3 60 10 5 25
V6S4 60 10 5 25
  • 37C 1hr>75C 10min>Etoh Precipitation

EtOH Precipitation[edit]

  • add 1ul of glycoblue
  • add 10ul of 3M NaoAc
  • add 250ul of 100% EtOH
  • Freeze the tube in -80C for 30min
  • centrifuge at 10000rpm for 20min, 4C
  • no pellet seen after centrifuge, added 1ul extra glycoblue and freeze for 1 hour, will spin at 14000 rpm for 30 min
  • doble the volume of all etoh precipitation reagents and spin again, this time we are able to see a blue pellet
  • remove supernatant and add 500ul of chilled 75% ETOH and transfer the DNA pellet to a 1.5ml tube (5000ul-3750ul100% EtOH + 1250ul ddh2o)
  • spin at 10,000rpm for 5min
  • dry the pellet in the hood and resuspend w 100ul ddh2o

TBU Gel quantification[edit]

File:ZhangLab 2 2013-02-14 18hr 12min.jpg

Name Type Volume          
    INT*mm2 Subtract Background Amount (ng) Dilution Actual Concentration (ng/ul) Actual Amount (ng)
U1 100BP_1.5ng 17252.4105141619 8238.9702455405 1.5      
U2 200BP_3ng 19505.830581319 11897.6403545773 3      
U3 100BP_0.75ng 9267.2702761862 3837.1701143566 0.75      
U4 200BP_1.5ng 12732.8203794676 6602.3301967648 1.5      
U5 V4S1 11703.1303487805 3537.3501054213 0.572788436370277 0.05 11.46 74.46
U6 V4S2 19698.1905870518 9345.380278514 2.14931306069451 0.05 42.99 279.41
U7 V4S3 28173.7208396423 16057.7104785571 3.97129978107829 0.05 79.43 516.27
U8 V4S4 14259.2804249597 6477.480193044 1.37085376036386 0.05 27.42 178.21
U9 V6S1 23770.1507084057 13991.6304169831 3.41048552980481 0.05 68.21 443.36
U10 V6S2 18602.5905544004 9715.6102895478 2.24980784320488 0.05 45.00 292.48
U11 V6S3 31858.4109494546 17246.710513992 4.29404050010375 0.05 85.88 558.23
U12 V6S4 48582.591447874 28414.9908468327 7.32554488822642 0.05 146.51 952.32