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===Verify 20 gap Probe Capture=== *Repeat probe capture from [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-28 2013-6-28] due to low capture efficiency of 20 gap probes and peculiar result for NTC reaction *Capture NTC, gDNA, and cDNA with Agi26k_20gap probes and gDNA with [http://genome-tech.ucsd.edu/LabNotes/index.php/CES36k18bp CES36k18bp] as positive control **[http://genome-tech.ucsd.edu/LabNotes/index.php/Capture_Test_of_CES36k18bp Capture Test of CES36k18bp] ====Synthesis of cDNA from 1ug Human Brain Ref RNA==== * Followed NEB E6300S protocol: [https://www.neb.com/protocols/1/01/01/first-strand-cdna-synthesis-e6300] * Used 1 ul of Human Brain Reference RNA at concentration 1ug/ul for cDNA synthesis * Mixed components in 0.2 ml microfuge tube labelled HBRR 1ug cDNA {| {{table}} | align="center" style="background:#f0f0f0;"|''' Components ''' | align="center" style="background:#f0f0f0;"|''' Volume ''' |- | RNA||1 ul (1 ug) |- | d(T)23VN (50 Β΅M)||2 ul |- | H2O||5 ul |- | Total||8 ul |} * Denatured RNA for 5 min at 70C and then put on ice * Added the following to tube {| {{table}} | align="center" style="background:#f0f0f0;"|''' Components ''' | align="center" style="background:#f0f0f0;"|''' Volume ''' |- | M-MuLV Reaction Mix||10 ul |- | M-MuLV Enzyme Mix||2 ul |- | Total||20 ul |} * Incubated for one hour at 42C * Inactivated enzyme for 5 min at 80C *Followed NEB E6111S protocol: [https://www.neb.com/protocols/1/01/01/second-strand-cdna-synthesis-e6111] *Added 48 ul H20 *Added 8ul 10x Second strand synthesis reaction buffer *Added 4ul Second strand synthesis enzyme mix *Mixed by pipetting *Incubated for 2.5 hours at 16C *Purified using Qiagen minelute column (eluted 21ul) *Measured DNA conc with Nandrop: 47ng/ul **19.5 ul (916.5 ng) sample left in tube ===Probe Capture=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Agi26k_20gap (16.79ng/ul)''' | align="center" style="background:#f0f0f0;"|'''CES36k18bp (75.5ng/ul)''' | align="center" style="background:#f0f0f0;"|'''''' |- | Probe Size||12964||36456|| |- | gDNA 12878 (100ng/ul)||300 ng||300 ng|| |- | gDNA MW||1.95x10^12 g/mol||1.95x10^12 g/mol|| |- | gDNA||1.5385x10^-19 mol||1.5385x10^-19 mol|| |- | Probe Required (1000:1)||1.5385x10^-16 mol||1.5385x10^-16 mol|| |- | Probe MW||8.1419x10^8 g/mol||1.1877x10^9 g/mol||(Probe size x (Probe length x 325 Da/nt + 79Da) |- | Amount Probe Required||125 ng (7.44ul)||183 ng (2.42 ul)|| |- | |} 20 gap probes from [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-4 2013-6-6] <br> {| {{table}} | align="center" style="background:#f0f0f0;"|'''Tube #''' | align="center" style="background:#f0f0f0;"|'''Probe Type''' | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Probe (ul)''' | align="center" style="background:#f0f0f0;"|'''10X Ampligase Buffer''' | align="center" style="background:#f0f0f0;"|'''Target (ul)''' | align="center" style="background:#f0f0f0;"|'''H2O''' | align="center" style="background:#f0f0f0;"|'''Total''' |- | 1||Agi26k_20gap||NTC||7.44||3||0||19.56||30 |- | 2||Agi26k_20gap||gDNA 12878||7.44||3||3||16.56||30 |- | 3||Agi26k_20gap||cDNA||7.44||3||19||0.56||30 |- | 4||CES36k18bp||gDNA 12878||2.42||3||3||21.58||30 |} '''Program'''<br> * 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h * -> add 2ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP mix) * -> 55 C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. ====SLN mix==== *Noi made the mix (NEED TO UPDATE THIS SECTION WHEN DETAILS ARE UP ON NOI'S WIKI) **Noi heat activated the Taq polymerase before adding to the mix {| class="wikitable" style="text-align:center;{{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Stock conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Final conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Prepare volume 10ul''' |- | AmpLigase||5||U/ul||0.5||U/ul||1.00 |- | 10x AmpLigase Buffer||10||x||1||x||1.00 |- | dNTP Mix||1||mM||100||uM||1.00 |- | Titanium Taq DNA||10||U/ul||2||U/ul||2.00 |- | H2O||||||||||5.00 |- | Total||||||||||10.00 |}
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