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>Alan6017518 (Created page with "==Expansion PCR== {| class="wikitable" <hiddentext>generated with de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion V1.8<\hiddentext> |- style="font-size:12pt" ...") |
>Alan6017518 |
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[https://www.dropbox.com/s/qjw1vc57tmzxnyl/Probe%3A2013-2-1%20-%20ZhangLabWiki.xlsx] | |||
==Expansion PCR== | ==Expansion PCR== | ||
*We will set up 8 reactions for each primer-oligo set (8 in total) | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
Line 136: | Line 138: | ||
| align="center" | 800 | | align="center" | 800 | ||
| align="center" | 800 | | align="center" | 800 | ||
|} | |||
95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 16 -> 72C 2min -> 15C hold. | |||
==QPCR Results== | |||
==EtOH Precipitation== | |||
*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== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="background-color:#008000;font-size:12pt" align="center" valign="bottom" | |||
| width="125" height="15" | Probe Set | |||
| width="67" | ng/ul | |||
| width="65" | ul | |||
| width="65" | ng | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V4S1 | |||
| align="center" | 27 | |||
| align="center" | 80 | |||
| align="center" | 2160 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V4S2 | |||
| align="center" | 47.7 | |||
| align="center" | 80 | |||
| align="center" | 3816 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V4S3 | |||
| align="center" | 61.6 | |||
| align="center" | 80 | |||
| align="center" | 4928 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V4S4 | |||
| align="center" | 35.9 | |||
| align="center" | 80 | |||
| align="center" | 2872 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V6S1 | |||
| align="center" | 61.2 | |||
| align="center" | 80 | |||
| align="center" | 4896 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V6S2 | |||
| align="center" | 49.7 | |||
| align="center" | 80 | |||
| align="center" | 3976 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V6S3 | |||
| align="center" | 74.9 | |||
| align="center" | 80 | |||
| align="center" | 5992 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V6S4 | |||
| align="center" | 86.7 | |||
| align="center" | 80 | |||
| align="center" | 6936 | |||
|} | |||
==DpnII Digestion== | |||
*We will setup a small testing reaction to optimize the restriction endonuclease reaction | |||
*I will test the reaction with 1ug of probes | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="background-color:#3366FF;font-size:12pt" | |||
| width="125" height="15" align="center" valign="bottom" | Probe Set | |||
| width="67" align="center" valign="bottom" | ng/ul | |||
| width="65" valign="bottom" | To get 1ug | |||
| width="65" valign="bottom" | 10X DpnII Buffer | |||
| width="65" valign="bottom" | DpnII | |||
| width="65" valign="bottom" | H2O | |||
| width="65" valign="bottom" | Total | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V4S1 | |||
| align="center" | 27 | |||
| align="center" | 37.04 | |||
| align="center" | 5 | |||
| align="center" | 1 | |||
| align="center" | 6.96 | |||
| align="center" | 50 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V4S2 | |||
| align="center" | 47.7 | |||
| align="center" | 20.96 | |||
| align="center" | 5 | |||
| align="center" | 1 | |||
| align="center" | 23.04 | |||
| align="center" | 50 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V4S3 | |||
| align="center" | 61.6 | |||
| align="center" | 16.23 | |||
| align="center" | 5 | |||
| align="center" | 1 | |||
| align="center" | 27.77 | |||
| align="center" | 50 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V4S4 | |||
| align="center" | 35.9 | |||
| align="center" | 27.86 | |||
| align="center" | 5 | |||
| align="center" | 1 | |||
| align="center" | 16.14 | |||
| align="center" | 50 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V6S1 | |||
| align="center" | 61.2 | |||
| align="center" | 16.34 | |||
| align="center" | 5 | |||
| align="center" | 1 | |||
| align="center" | 27.66 | |||
| align="center" | 50 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V6S2 | |||
| align="center" | 49.7 | |||
| align="center" | 20.12 | |||
| align="center" | 5 | |||
| align="center" | 1 | |||
| align="center" | 23.88 | |||
| align="center" | 50 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V6S3 | |||
| align="center" | 74.9 | |||
| align="center" | 13.35 | |||
| align="center" | 5 | |||
| align="center" | 1 | |||
| align="center" | 30.65 | |||
| align="center" | 50 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V6S4 | |||
| align="center" | 86.7 | |||
| align="center" | 11.53 | |||
| align="center" | 5 | |||
| align="center" | 1 | |||
| align="center" | 32.47 | |||
| align="center" | 50 | |||
|} | |||
*Incubate reaction at 37C for 1 hour followed by 20min at 65C for heat inactivation | |||
*Column purification, elute with 30ul EB | |||
==PAGE Gel== | |||
[[File:ZhangLab_2 2013-02-04 14hr 19min.jpg|400px]] | |||
==DpnII Digestion for remaining probes== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="background-color:#95B3D7;font-size:12pt" align="center" valign="bottom" | |||
| width="125" height="15" | Probe Set | |||
| width="67" | ng/ul | |||
| width="100" | Remaining Volume | |||
| width="101" | 10X DpnII Buffer | |||
| width="65" | DpnII | |||
| width="65" | H2O | |||
| width="65" | Total | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V4S1 | |||
| align="center" | 27 | |||
| align="center" | 42.96 | |||
| align="center" | 10 | |||
| align="center" | 5 | |||
| align="center" | 42.04 | |||
| align="center" | 100 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V4S2 | |||
| align="center" | 47.7 | |||
| align="center" | 59.04 | |||
| align="center" | 10 | |||
| align="center" | 6 | |||
| align="center" | 24.96 | |||
| align="center" | 100 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V4S3 | |||
| align="center" | 61.6 | |||
| align="center" | 63.77 | |||
| align="center" | 10 | |||
| align="center" | 7 | |||
| align="center" | 19.23 | |||
| align="center" | 100 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V4S4 | |||
| align="center" | 35.9 | |||
| align="center" | 52.14 | |||
| align="center" | 10 | |||
| align="center" | 6 | |||
| align="center" | 31.86 | |||
| align="center" | 100 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V6S1 | |||
| align="center" | 61.2 | |||
| align="center" | 63.66 | |||
| align="center" | 10 | |||
| align="center" | 7 | |||
| align="center" | 19.34 | |||
| align="center" | 100 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V6S2 | |||
| align="center" | 49.7 | |||
| align="center" | 59.88 | |||
| align="center" | 10 | |||
| align="center" | 6 | |||
| align="center" | 24.12 | |||
| align="center" | 100 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V6S3 | |||
| align="center" | 74.9 | |||
| align="center" | 66.65 | |||
| align="center" | 10 | |||
| align="center" | 7 | |||
| align="center" | 16.35 | |||
| align="center" | 100 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | V6S4 | |||
| align="center" | 86.7 | |||
| align="center" | 68.47 | |||
| align="center" | 10 | |||
| align="center" | 7 | |||
| align="center" | 14.53 | |||
| align="center" | 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 | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="background-color:#F79646;font-size:12pt" | |||
| width="125" height="15" align="center" | Probe Set | |||
| width="124" valign="bottom" | Concentration (ng/ul) | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | V4S1 | |||
| align="center" | 20.5 | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | V4S2 | |||
| align="center" | 40.7 | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | V4S3 | |||
| align="center" | 57.7 | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | V4S4 | |||
| align="center" | 32.5 | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | V6S1 | |||
| align="center" | 53 | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | V6S2 | |||
| align="center" | 45 | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | V6S3 | |||
| align="center" | 71.2 | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | V6S4 | |||
| align="center" | 83.1 | |||
|} | |||
==Lambda exonuclease digestion== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="background-color:#CCFFCC;font-size:12pt" align="center" | |||
| width="125" height="15" | Probe Set | |||
| width="124" | Volume | |||
| width="120" | 10X Lambda Exo Buffer | |||
| width="110" | Lambda Exo (10U/ul) | |||
| width="65" | H20 | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | V4S1 | |||
| align="center" | 60 | |||
| align="center" | 10 | |||
| align="center" | 5 | |||
| align="center" | 25 | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | V4S2 | |||
| align="center" | 60 | |||
| align="center" | 10 | |||
| align="center" | 5 | |||
| align="center" | 25 | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | V4S3 | |||
| align="center" | 60 | |||
| align="center" | 10 | |||
| align="center" | 5 | |||
| align="center" | 25 | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | V4S4 | |||
| align="center" | 60 | |||
| align="center" | 10 | |||
| align="center" | 5 | |||
| align="center" | 25 | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | V6S1 | |||
| align="center" | 60 | |||
| align="center" | 10 | |||
| align="center" | 5 | |||
| align="center" | 25 | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | V6S2 | |||
| align="center" | 60 | |||
| align="center" | 10 | |||
| align="center" | 5 | |||
| align="center" | 25 | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | V6S3 | |||
| align="center" | 60 | |||
| align="center" | 10 | |||
| align="center" | 5 | |||
| align="center" | 25 | |||
|- style="font-size:12pt" align="center" | |||
| height="15" | V6S4 | |||
| align="center" | 60 | |||
| align="center" | 10 | |||
| align="center" | 5 | |||
| align="center" | 25 | |||
|} | |||
*37C 1hr>75C 10min>Etoh Precipitation | |||
==EtOH Precipitation== | |||
*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== | |||
[[File:ZhangLab_2 2013-02-14 18hr 12min.jpg|400px]] | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- valign="bottom" | |||
| width="69" height="12" | Name | |||
| width="96" | Type | |||
| width="69" | Volume | |||
| align="center" width="122" | | |||
| align="center" width="69" | | |||
| align="center" width="69" | | |||
| align="center" width="112" | | |||
| align="center" width="105" | | |||
|- valign="bottom" | |||
| align="center" height="12" | | |||
| align="center" | | |||
| INT*mm2 | |||
| Subtract Background | |||
| Amount (ng) | |||
| Dilution | |||
| Actual Concentration (ng/ul) | |||
| Actual Amount (ng) | |||
|- valign="bottom" | |||
| height="12" | U1 | |||
| 100BP_1.5ng | |||
| align="center" | 17252.4105141619 | |||
| align="center" | 8238.9702455405 | |||
| align="center" | 1.5 | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
|- valign="bottom" | |||
| height="12" | U2 | |||
| 200BP_3ng | |||
| align="center" | 19505.830581319 | |||
| align="center" | 11897.6403545773 | |||
| align="center" | 3 | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
|- valign="bottom" | |||
| height="12" | U3 | |||
| 100BP_0.75ng | |||
| align="center" | 9267.2702761862 | |||
| align="center" | 3837.1701143566 | |||
| align="center" | 0.75 | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
|- valign="bottom" | |||
| height="12" | U4 | |||
| 200BP_1.5ng | |||
| align="center" | 12732.8203794676 | |||
| align="center" | 6602.3301967648 | |||
| align="center" | 1.5 | |||
| align="center" | | |||
| align="center" | | |||
| align="center" | | |||
|- valign="bottom" | |||
| height="12" | U5 | |||
| V4S1 | |||
| align="center" | 11703.1303487805 | |||
| align="center" | 3537.3501054213 | |||
| align="center" | 0.572788436370277 | |||
| align="center" | 0.05 | |||
| align="center" | 11.46 | |||
| align="center" | 74.46 | |||
|- valign="bottom" | |||
| height="12" | U6 | |||
| V4S2 | |||
| align="center" | 19698.1905870518 | |||
| align="center" | 9345.380278514 | |||
| align="center" | 2.14931306069451 | |||
| align="center" | 0.05 | |||
| align="center" | 42.99 | |||
| align="center" | 279.41 | |||
|- valign="bottom" | |||
| height="12" | U7 | |||
| V4S3 | |||
| align="center" | 28173.7208396423 | |||
| align="center" | 16057.7104785571 | |||
| align="center" | 3.97129978107829 | |||
| align="center" | 0.05 | |||
| align="center" | 79.43 | |||
| align="center" | 516.27 | |||
|- valign="bottom" | |||
| height="12" | U8 | |||
| V4S4 | |||
| align="center" | 14259.2804249597 | |||
| align="center" | 6477.480193044 | |||
| align="center" | 1.37085376036386 | |||
| align="center" | 0.05 | |||
| align="center" | 27.42 | |||
| align="center" | 178.21 | |||
|- valign="bottom" | |||
| height="12" | U9 | |||
| V6S1 | |||
| align="center" | 23770.1507084057 | |||
| align="center" | 13991.6304169831 | |||
| align="center" | 3.41048552980481 | |||
| align="center" | 0.05 | |||
| align="center" | 68.21 | |||
| align="center" | 443.36 | |||
|- valign="bottom" | |||
| height="12" | U10 | |||
| V6S2 | |||
| align="center" | 18602.5905544004 | |||
| align="center" | 9715.6102895478 | |||
| align="center" | 2.24980784320488 | |||
| align="center" | 0.05 | |||
| align="center" | 45.00 | |||
| align="center" | 292.48 | |||
|- valign="bottom" | |||
| height="12" | U11 | |||
| V6S3 | |||
| align="center" | 31858.4109494546 | |||
| align="center" | 17246.710513992 | |||
| align="center" | 4.29404050010375 | |||
| align="center" | 0.05 | |||
| align="center" | 85.88 | |||
| align="center" | 558.23 | |||
|- valign="bottom" | |||
| height="12" | U12 | |||
| V6S4 | |||
| align="center" | 48582.591447874 | |||
| align="center" | 28414.9908468327 | |||
| align="center" | 7.32554488822642 | |||
| align="center" | 0.05 | |||
| align="center" | 146.51 | |||
| align="center" | 952.32 | |||
|} | |} |
Latest revision as of 01:26, 16 February 2013
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 |