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==Probe Synthesis CES36K 18BP== ==Expansion PCR== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''X2.1''' |- | CES36K18BP (20nM)||1||2.1 |- | 2X iQ MasterMix||50||105 |- | 100uM AP1V6||0.4||0.84 |- | 100uM AP2V6||0.4||0.84 |- | H2O||48.2||101.22 |} 94C 2min -> (94C 30sec -> 54C 45sec -> 72C 45sec) x 15 -> 72C 3min -> 15C hold Purify the amplicons with two Qiaquick columns, eluted with 30ul EB. Yield: 10.3ng/ul x 60ul = 618ng Concentration = 10.3ng/ul / (140 * 660) = 108nM Take 20ul, add 196ul EB -> 10nM ==Production PCR== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''X33''' |- | 10nM 1st round amplicon||0.2||6.6 |- | 2X Kapa SYBR qPCR Master Mix||50||1650 |- | 100uM pAP1V61U||0.4||13.2 |- | 100uM AP2V6||0.4||13.2 |- | H2O||44||1452 |} 95C 30sec -> (95C 3sec -> 55C 30sec-> 60C 20sec) x 16 -> 60C 2min -> 15C hold. * I did a test run with one well and see how many cycles i need to run [[File:120420.JPG|400px]] *Looks like 12 cycles will be enough *Now I will go ahead and move on with the production {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume needed''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 100% ETOH||2.5x sample||8000 |- | 3M NaOAC||0.1x sample||320 |- | Glycoblue||1/300x sample||10.66666667 |- | sample||sample volume||3200 |} *Mix all content by repeat pipetting using multichannel pipette *Pool all 24 wells PCR product into tray using multichannel pipette(P300) *Mix all content by repeat pipetting using multichannel pipette *Aliquot about 3750uL to 2 x 15mL tube *Store in -80C for more than 20 mins or forever *Continue on Apr29 [[http://genome-tech.ucsd.edu/LabNotes/index.php/AlanFung:LabNotes/Probe/2012-4-29]] ==Production PCR== *Continue from Apr04 [[http://genome-tech.ucsd.edu/LabNotes/index.php/AlanFung:LabNotes/Probe/2012-4-4]] *Turn on Centrifuge fix temperature at 4C, place a tube of 75% ethanol into 4C fridge *Centrifuge frozen 15mL tubes at 3000rpm for 20m at 4C *Remove all liquid '''CAUTION: DO NOT DISCARD BLUE PELLET AT THE BOTTOM OF THE TUBE''' *Add in 750uL cold 75% Ethanol, mix by repeat pipetting and transfer to a 1.5mL tube *Centrifuge at 10,000rpm for 5min at 4C *Remove all liquid, air dry sample for 5min or until samples dried out *Resuspend the DNA with 100uL RNAse free H2O *Re-purify using 4 qiaquick tubes elute with 41ul EB *Yield 4x40x201ng/ul=32.16ug *Store in 4C, continue on Apr30 [[http://genome-tech.ucsd.edu/LabNotes/index.php/AlanFung:LabNotes/Probe/2012-4-30]] ==Nanodrop== ==Lambda exo digestion== * Split the libraries into half, 80ul each around 16.8ug {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Qiaquick amplified amplicon||80 |- | 10X lambda Exo Buffer||10 |- | Lambda Exo||10 |- | H2O||0 |- | Total||100 |} *Incubate at 37C for one hour *Purify with 4xQiaquick elute with 31ul EB (since each qiaquick column max binding capacity is 10ug) *Yield ssDNA: 75.5ng/ul x 120ul = 9.06ug *Labeled tube and stored @ -20C *Continue on 5/1/2012 [[http://genome-tech.ucsd.edu/LabNotes/index.php/AlanFung:LabNotes/Probe/2012-5-1]] ==Removal of amplification adaptors== *To 75ul ss-amplicon (5.7ug) , add 5ul USER enzyme, 37C 1h; *Add 10ul 10x Dpn II buffer, 5ul 100uM RE-DpnII-V6 guide oligo, 94C 2min -> 37C 3min -> add 5ul Dpn II -> 37C 2h *add 5ul USER, 10ul 10x buffer, 85ul H2O *-> 37C 2h -> 75C 20min -> purify with two Qiaquick column *Continue May 2 [[http://genome-tech.ucsd.edu/LabNotes/index.php/AlanFung:LabNotes/Probe/2012-5-2#Gel_Quantification]] ==Gel Quantification== *Elute with 120ul EB and used 1ul to run gel [[File:ZhangLab_2 2012-05-02 16hr 03min.jpg|300px]] *Digestion didn't complete *Talked to Noi and she thinks so too *Will work together with Noi on 5/7 and repeat the digestion protocol. ==Troubleshooting May7== *Noi and I think that the probes are not digested completely ==USER Digestion== *from the 75.5ng/ul tube, take 40ul product add 2.5ul USER enzyme *Incubate at 37C for 1.5hr and run a denaturing gel *Since I ran a TBE gel on my end product from last week, I will rerun on a denaturing gel so that the 2nd structure wont be the reason why we see the larger than expected DNA size [[File:ZhangLab_2 2012-05-07 13hr 31min.jpg|250px]] *Turns out the end product is at the expected size, it was the TBE gel leading to larger than expected DNA size. *Will move on with the tube we started today and pool it with the end product and perform a size selection for both tubes. ==Page Denaturing Gel== *ssDNA nanodrop result:28.8ng/ul x 150ul = 4.320ug *Limit for 2D gel is around 1ug I will run 4 gels *Prepare a 2D well denaturing gel by scraping off the gel residue outside and peel of the sticker at the back of gel *Load 4 gels into the gel running module *Add in fresh 0.5X TBE buffer to the inside core full the outer core up to the metal line *Pre-run the gel for 30 min at 200V *Dilute sample to no more than 40ng/uL *Dilute TBE-Urea sample buffer (2X) to 1X in each tube. For 4 gels; Mix 0.5 uL Invitrogen 10bp ladder with 9.5 uL ddH2O, 10uL 2X TBE-Urea sample buffer *Denature samples and ladder at 75C for 7 min, then quickly transfer to cooling rack, wait for 5 mins *Flush the wells very well with P1000 & P200 pipette to remove all urea. '''NOTE: DO NOT TOUCH THE TUBE WITH YOUR HANDS IN THE FOLLOWING STEP, GRAB THE CAP''' *Load the samples into the wells, run at 200V for 30 min. *Stain gel with 4uL SYBR Gold in ~100 mL of TBE in clean tray. *Place tray on an orbital shaker for ~5 min. ==Purify the product from the gel== *Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use G20 needle to punch a small hole at the bottom of the 0.5 mL tube. Use two assemblies for each PAGE gel. *Cut out the correct band (85-92 nt in this case), chop the band into small slices, split into two half and transfer each half to one shearing assembly. *Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube. *Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 37C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight. *Spin down all tubes at 13,000rpm for 2min *In the PCR hood, transfer the liquid layer of the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer supernatant to 1.5mL tube. ==Quantification of DNA using denaturing gel and a quantitative ladder== Combine all tubes into a 1.5mL tube 1. Add 1ul of low mass ladder, 4ul of H2O and 5 uL of 2x TBE-Urea buffer 1. Add 2ul of low mass ladder, 3ul of H2O and 5 uL of 2x TBE-Urea buffer 1. Add 4ul of low mass ladder, 1ul of H2O and 5 uL of 2x TBE-Urea buffer 2. Dilute sample 10x (1uL sample + 9 uL of water) so total is 10uL of sample, and add 10 uL of 2x TBE buffer 2. Dilute sample 10x (2uL sample + 8 uL of water) so total is 10uL of sample, and add 10 uL of 2x TBE buffer 3. Denature the DNA with loading dye on thermalcycler 75C for 7 min, quickly transfer to cooling rack, wait for 1 min. 4. Use 6% 10 well TBU gel, add 0.5x TBE buffer, flush the wells very well to remove all urea. 5. Pre-run the gel at 200V for 10 min, then load the samples into the wells, run at 200V for 30 min. Load the ladder as 8, 4, 2, 1 uL, and load the samples as 10, 5, 2.5 uL. 6. Stain the gel with 5 uL SYBR gold in ~100 mL of TBE in clean tray. Place the tray on an orbital shaker for 5 min. Then quantify the gel to calculate the % yield. [[File:ZhangLab_2 2012-05-14 15hr 46min.jpg|400px]] *Using the linear trendline we obtain the following formula, where x is ng and y is the intensity *y = 546.85x + 2758.3 *the 1ul probe's intensity is at 18876.28 which converts to 29.47ng *the 2ul probe's intensity is at 22667.29 which converts to 36.41ng
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