AlanFung:LabNotes/Probe/2013-2-1
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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 |