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===Page Size Selection of 0 gap probes=== USER/DpnII digested 0 gap probes: Noi made 11.36ug/~400ul [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-29 2013-5-29 ] I made 10.58ug/180ul [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-16 2013-5-16 ] <!--If Combined: 11.36ug + 10.58ug / (410ul + 180ul) = 21.94ug/590ul = 37.19ng/ul (590ul)--> To run ~3ug/gel, I use ~100ul of probes that Noi made for each of the 4 gels * Ran TBU gel at 220V for 30 min with samples and ladder prepared below ** Heated TBE buffer in microwave ** Denatured samples/ladder for 10 min at 75C before putting on cold rack for 1 min ** Size selected for brightest band below 200bp {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' | align="center" style="background:#f0f0f0;"|'''4X Volume''' |- | Enzyme digested probes (~11.36/400ul)||106||424 |- | TBE-Urea Buffer 2X||106||424 |- | H2O||0||0 |- | Total||212||848 |- | |} {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' | align="center" style="background:#f0f0f0;"|'''4X Volume''' |- | Low MW DNA ladder (1 ug/ul)||1||4 |- | TBE-Urea Buffer 2X||5||20 |- | H2O||4||16 |- | total||10||40 |- | |} [[File:2013-06-26_Agi26k_0gap_SizeSelect1.jpg | 450px]] [[File:2013-06-26_Agi26k_0gap_SizeSelect2.jpg | 450px]] [[File:2013-06-26_Agi26k_0gap_SizeSelect3.jpg | 450px]] [[File:2013-06-26_Agi26k_0gap_SizeSelect4.jpg | 450px]] *Put cut out gels in 0.5mL tube with holes at bottom in 2 mL tubes (6 tubes for 4 gels) *Centrifuged the tubes at 15,000 rpm, for 3 min at RT. *Transfered the gel remaining in 0.5 mL tube to 2 mL tube below with pipette tip. *Added 675 ul of 1X TE buffer (because 1.5x more gel compared to normal protocol using 1.5mL tube) *Vortexed for 45-60 min at 37 C in incubator *Centrifuged at 15,000 rpm for 3 min at RT *Transfered the clear spnt. to Nanosep column and centrifuged at 15,000 rpm for 3 min *Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min *Transfered spnt to fresh 1.5 mL tube (volume ~ 500ul/ tube) *Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2 *Vortexed and placed the 8 tubes at -80C overnight (had to use 8 because volume was too much for 6) Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-27 ====First Strand cDNA synthesis==== * Followed NEB E6300S protocol: [https://www.neb.com/protocols/1/01/01/first-strand-cdna-synthesis-e6300] Got 2ul aliquot of Human Brain Reference RNA from Blue at concentration 1ug/ul * Mixed components in two 0.2 ml microfuge tubes labelled HBBR 1ug cDNA {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''2X Volume''' |- | RNA||1 ul (1 ug)||2 ul (2 ug) |- | d(T)23VN (50uM)||2 ul||4 ul |- | H2O||5 ul||10 ul |- | Total||8 ul||16 ul |- | |} * Denatured RNA for 5 min at 70C and then put on ice ** Tightened the thermocycler too much and bent the tube slightly out of shape * Added the following to each 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 * Kept on ice ====Second Strand cDNA synthesis==== *Follow NEB E6111S protocol: [https://www.neb.com/protocols/1/01/01/second-strand-cdna-synthesis-e6111] *Added 48 ul H20 to each tube *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 23ul into 1.5ml tube labelled HBRR cDNA) *Measured DNA conc with Nandrop: **43ul at 63.7ng/ul (2.74 ug) sample left in each tube **"High yield" because RNA was not digested and went from single stranded to double stranded; slightly higher but consistent with last time [http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-3-9 2013-3-9]
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