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AlanFung:LabNotes/Reprogramming/2011-10-7
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==Amplify POLR1D Gene== ==Material== *Total RNA (1ug/ul) (Clontech-from Rui) *GSP-Gene specific primer *1 step RT-PCR Kit ==Primer Design== *I received this pair of primers from Sergio CGG(RE)ATGGAAGAGGATCAGGAGCTGGAGAG (Forward) 5β-3β CGG(RE)CTAGAATGTGGATTCATTTCTGCTTGC (Reverse) 5β-3β *I am not going to add restriction enzyme, and hence I will just order the primers without the RE sites. *After blasting the primers o NCBI blast the the primers matched 100% to POLR1D gene when cgg is removed from 5' end *Order from IDT after confirming with Kun ATGGAAGAGGATCAGGAGCTGGAGAG (Forward) 5β-3β CTAGAATGTGGATTCATTTCTGCTTGC (Reverse) 5β-3β *After blasting the primers aligns at transcript NM_015972.3 this locus is 817bp long, with primer 3 the product size is 402 with Tm at 67.46C and 63.40C (avg~65C)for forward and reverse primer respectively. I will use annealing temperature at 55C ==cDNA Synthesis & RT-PCR with Superscript III== ===Some parameters to be considered=== *Starting material 10ng total RNA *Preheat thermal cycler to 45-60C depending on the temperature selected for cDNA synthesis (Protocol recommends 30min incubation at 55C as a starting point) *For high gc content templates use a 60C cDNA synthesis temperature *cDNA synthesis can be accomplished in a 15-30min incubation at 45-60C. For small targets and incubation time of 5min may be sufficient *Annealing temperature should be 10C below the melting temperature of the primers used *Extension time varies with the size of the amplicon (approximately 1 min per 1kb of amplicon) *For all targets up to 4.5kb, 2ul of superscript III RT/Platinum Taq Mix is sufficient ===Protocol=== *Dilute primers to 10uM (1:10) *Dilute RNA to 10ng/ul (1:100) *Preheat thermal cycler to 55C *Prepare Reaction on ice (Setup two reactions, 1 with 10ng total RNA, one with 1ul ddH2O as negative control) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume per reaction''' | align="center" style="background:#f0f0f0;"|'''MM''' |- | 2X reaction mix||25||55 |- | Template RNA (10ng/ul)||1||Do not add to MM |- | Sense primer (10uM)||1||2.2 |- | Anti-sense primer (10uM)||1||2.2 |- | Superscript III RT/Platinum Taq Mix||2||4.4 |- | ddH2O||20||44 |- | Total||50||110 |} *Run scdna1s 55C 30min 94C 2min 40x {94C 15s 62C 30s 68C 1min} 68 5min ===Purification=== *Perform Qiaquick purification ===PAGE Analysis=== [[File:ZhangLab_2 2011-10-18 11hr 42min.jpg|300px]] ===Perform Cloning and Sanger Sequencing=== ===Blunting with NEB end repair kit=== *Quantify the product-Qubit 40.4ng/ul *Starting material should be 1-5ug *Take of PCR product {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''POLR1D''' |- | Fragmented DNA||24.8 |- | 10X End Repair Reaction Buffer ||10 |- | End Repair Enzyme||5 |- | H2O||60.2 |- | Total Volume||100 |} *Incubate at 20C for 30min *Minelute with 10ul EB ===Qubit=== 20.8ng/ul ===Ligating into pCR blunt vector=== **Calculate the amount of PCR product needed for ligation *Talked to Rui and she used 5ng vector, I will try the same protocol as I got too many colonies from the previous cloning, however I will maintain insert to vector ratio at 10:1 **x ng insert = (10*500*5/3500) **X=7.14ng needed *Make 5ng/ul vector (Mix 2ul vector with 8ul water) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''POLR1D''' |- | PCR Blunt (5ng/ul)||1 |- | Blunt PCR Product||0.4 |- | 10X ligation buffer (with ATP)||1 |- | Sterile water||6.6 |- | T4 DNA Ligase||1 |- | Total Volume||10 |} *Incubate at 16C for 1 hr ===Heat-shock transfection=== *Equilibrate water bath to 42C. *Bring the SOC medium, LB plates (w/ antibiotics - Kanamycin 50ug/mL) to RT for 30min. *Thaw the TOP10 E.coli competent cell vial on ice and split 25ul to a new 1.5ml tube *Transfer '''1uL''' ligation reactions into the vial of competent cells. Mix by gentle stiring using tips. ('''Mixing by repeat pipetting is prohibited in the protocl'''). *Set up conpetent cell vials in the foam-board holder(for water bath). Incubate the vials on ice for 30 mins. *Heat shock the cells for 30 seconds at 42 C without shaking. Immediately transfer the vials on ice for 2min. *Add 125ul of SOC medium into each vial (drop the SOC slution slowly throw the tube wall). *Shake the vials horizontally at 37C for 1 hour using vortex in the incubator at the lowest speed. *During the 1hr incubation. *After 1hr shacking mixing, plate '''50uL''' from each transformation vial on agar plates (w/ Kanamycine and xgal) *Incubate plates at 37C for about 20hr. ==Preparation for Sanger Sequencing== ===PCR amplification and preparation for sanger sequencing=== *Add 12.5ul of 100ul of F and R primer and add 475ul sterile h2o to make 2.5uM primer mix {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume per reaction''' | align="center" style="background:#f0f0f0;"|'''MM''' |- | DNA||Pick one colony per well||Do not add to MM |- | H2O||25||440 |- | M13-21 F+R Primer (2.5uM each)||8||140.8 |- | 2X Taq MM||15||264 |- | Total||48||N/A |} *Add 48ul MM to each colony 94C 10 min to break up the cells 94C 30 sec -> (94C for 30 sec, 60C for 30 sec, 72C for 1 min) x30 -> 72C 5 min ->15C forever ===PAGE===
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