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Dinh:Protocols/Probes Prep Nov24
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=November 25, 2009= ==Step 1: PCR Amplification == Reaction Mix (100ul reaction volume): {| border="1" !Reagent !Final Conc. !Vol (1x) !Vol (8x) !Vol (100x) |- |ddH20 | |49.4ul |395ul |4940ul |- | 2x KAPA qPCR Master Mix |1x |50ul |400ul |5000ul |- |AP1V4IU (100uM) |0.3uM |0.3ul |2.4ul |30ul |- |AP2V4 (100uM) |0.3uM |0.3ul |2.4ul |30ul |- |Template CpG30k (20nM) |33pg |0.05ul |0.4ul |5ul |- !Total | |100.05ul |800.4ul |10005ul |} Program 94C - 30 s 94C - 3 s 60C - 30 s Plate Read Cycle to 2 for 29 more times 72C - 5 min 15C - forever end Protocol: Place KAPA 2x qPCR MM on ice. Add ddH20, primers, and template to 15ml tube (RNAase/DNAase free). Vortex & leave on ice for 2-3 min Prepare strip tubes on cooling rack Add KAPA 2x MM & vortex, shake down reagents, then pour into 55ml solution basin Use multichannel p-100 pipettor. Pipette 101ul to each tube. For last row, use p-200 to pipette 101ul to each tube. Place caps on strip tubes Spin pairs of strip tubes for 5-10 s to remove bubbles Prepare new run on Chromo4 RT-PCR machine, let it get to 94 C Pause program, place tubes in machine, and resume run 3. Stopped at 19 cycles. ==Notes:== 8x reaction mix was prepared as test run. 100ul was aliquoted to 6 tubes and amplified using RT-PCR for 26 cycles (over-amplified), amplification slope started at cycle 13 and plateaued at cycle 25. For the rest of the reaction mix, 25ul was aliquoted to 8 tubes. Only 4 tubes were amplified using the the same program for 26 cycles (over-amplified), amplification sloped started also at cycle 13 and plateaued at cycle 25. Concluded no difference between 25ul and 100ul reaction volumes. The leftover 4 tubes of 25ul reaction volume was used in another run: Program 95C - 30 s 95C - 3 s 60C - 30 s 72C - 3 s Plate Read Cycle to 2 for 29 more times 72C - 5 min 15C - forever end Recommended by KAPA qPCR manual: 95C denaturation (initital & in cycle), and 72C during plate read. However, for this run, amplification slope started at cycle 15 and plateaued also at cycle 23. Concluded that other program used was better suited for this probe set. ==Step 2: Ethanol Precipitation== Performed as in previous protocols using 8x15ml tubes. ==Step 3: Lambda Exonuclease== All dissolved products in RNase free dH20 was pooled. 130ul sample, 15ul 10x lambda exonuclease buffer, 5ul lambda exonuclease. 8 reactions. ==Step 4: QiaQuick Purification== Performed as in previous protocols. All reactions were first pooled and 150 ul was aliquoted to 7 tubes containing 750ul buffer PE. Divided solution equally to two columns. Tested EB vs DNase Free H20 ~130ul was aliquoted to 1 tube containing 750ul buffer PBI. Divided solution equally to two columns. 1 column was eluted with elution buffer (EB). 1 column was eluted with DNase free dH20. ==Step 5: USER Digestion== Performed as in previous protocols. Digested for 3 hours. 3ul USER Enzyme 7ul 10x DpnII buffer ==Step 6: DpnII Digestion== 3ul 100nM DpnII oligo 1ul 10x DpnII buffer ==Step 7: PAGE Size selection== Performed as in previous protocols. Pooled all reactions. 100ul sample + 100ul loading dye per 2D well, 6x2D gels. Concentration of ssDNA in solution was 50.9ng/ul x 100ul loaded = 5090ng loaded per 2D well. [[Image:ZhangLab 2 2009-12-02 ss kapa set1-2.jpg|300px|thumb|center| Gels 1-2]] [[Image:ZhangLab 2 2009-12-02 ss kapa set3-4.jpg|300px|thumb|center| Gels 3-4]] [[Image:Dinh-ZhangLab 2 2009-12-02 ss kapa set5-6.jpg|300px|thumb|center| Gels 5-6]] ==Step 8: EtOH ppt== Pooled all flowthrough and divide equally to 10x1.5mL tubes. Then during washing step, combing blue pellets to one tube *Since all tubes are expected to have approx. the same DNA mass, if no blue pellet is seen: add 0.5ul Glycoblue, vortex, and re-freeze solution at -70C for 20min. ==Step 9: Quantification== [[Image:Dinh-ZhangLab_2_2009-12-02_band_check_CpG30K_kapa_amplified_probes_v2.jpg|300px|thumb|left|Bands check]] {| {{table}} border = "1" | align="center" style="background:#f0f0f0;"|'''Lane''' | align="center" style="background:#f0f0f0;"|'''STEP''' | align="center" style="background:#f0f0f0;"|''' Volume Loaded(ul)''' | align="center" style="background:#f0f0f0;"|'''Qubit Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Dilution''' | align="center" style="background:#f0f0f0;"|'''Mass Loaded''' |- | 1||After EtOH ppt||1||103||1||103 |- | 2||After Lambda Exonuclease||1||55.25||1||55.25 |- | 3||After QiaQuick||1||83.42||1||83.42 |- | 4a||After USER/DpnII||0.6||50.9||1||30.54 |- | 4b||After USER/DpnII||0.3||50.9||1||15.27 |- | W||After USER/DpnII(water diluted in last QQ step)||1||<50.9||1||- |- | E||After USER/DpnII(EB diluted in last QQ step)||1||<50.9||1||- |- |} # The use of EB does not negatively affect digestion or yield. ===Purification Table=== {| {{table}} border = "1" | align="center" style="background:#f0f0f0;"| | align="center" style="background:#f0f0f0;"|'''Step''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Purity''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''ssDNA (ng)''' | align="center" style="background:#f0f0f0;"|'''% Recovery''' |- | 1||After first EtOH ppt||1020||9.41||103||105060|| |- | 2||After lambda exon||1103||0.92||55.25||60941||58.01% |- | 3||After qiaquick||420||2.63||83.42||35035||57.49% |- | 4||After USER &DpnII||596||0.7||50.9||26522(*)||75.70% |- | 5||After size selection & Nanosep||4047||0.13||0.33||1315||4.96% |- | 6||After final EtOH ppt||20||202.350||23.48||470||35.70% |- |} # Purification factor: previous step volume/current step volume # % Recovery: previous step mass/current step mass # (*)Subtracted mass of oligo added: 100nM*4238g/moles*1/1000*3ul/rxn*9rxns = 3800ng ===Conclusions===
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