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=RCA Test (Started [[Daniel:Notebook/ComboLock/2017-3-24|March 24]])= [[Daniel:Notebook/ComboLock|Back to Calendar]] I'll try qPCR on the RCA product again, this time on the NEB 12 hr samples (since I am running low on X samples), but for few cycles and with only the forward primer (which should be RC to the RCA product. This will be fed into Bglii digestion <ol> <li>qPCR-Second strand synthesis</li> <ol type="A"> <li>Make the following 13.2X master mix</li> <ol type="a"> <li>13.2 uL AmpF-6.4Sol (10 uM)</li> <li>264 uL Kapa SYBR 2X Master Mix</li> <li>224.4 uL nfH2O</li></ol> <li>Aliquot 38 uL master mix into each lane</li> <li>Add 2 uL sample according to plate layout</li> [[Image:PlateLayout-20170403-RCATest-CPLP-ssSynthesis.png|600px]] <li>Run the following protocol</li> <ol type="a"> <li>95C for 2 min</li> <li>95C for 5 sec</li> <li>55C for 30 sec</li> <li>72C for 45 sec</li> <li>Goto (b) x20</li> <li>72C for 2 min</li></ol> </ol></ol> ==Protocol-[[Daniel:Notebook/ComboLock/2017-3-29|Post-Bglii samples]]== <ol start="2"> <li>TBE Gel</li> <ol type="A"> <li>Mix 80 uL TBE and 20 uL 6X dye</li> <li>Aliquot 10 uL on to parafilm per sample/ladder</li> <li>Add 2.5 uL reaction or 1.5 uL ladder to appropriate aliquot</li> <li>Add 10 uL mix to gel lanes</li> <li>Run gel for 24 minutes at 230V</li> <li>Stain with 2 uL SYBR gold for 3 minutes</li> <li>Rinse and image in gel doc</li> </ol> <li>Qiaquick Column</li> <ol type="A"> <li>Add 200 uL (5X) PB buffer to 40 uL sample</li> <li>Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through</li> <li>Add 700 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through</li> <li>Dry spin column for 1 minute at 14000 rpm; discard flow through</li> <li>Let stand with cover open in fume hood for ~5 minutes</li> <li>Transfer column to a new 1.5 mL eppendorf tube</li> <li>Add 35 uL nfH2O to column</li> <li>Let stand 1 minute</li> <li>Spin for 1 minute at 14000 rpm</li> </ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 40 uL 10X TBE and 20 uL 6X dye</li> <li>Aliquot 10 uL on to parafilm per sample, and 10 uL TBE ladder</li> <li>Add 6 uL reaction or 1.5 uL ladder to appropriate aliquot</li> <li>Add 10 uL mix to gel lanes</li> <li>Run gel for 24 minutes at 230V</li> <li>Stain with 2 uL SYBR gold for 3 minutes</li> <li>Rinse and image in gel doc</li> </ol></ol> ===Results=== <gallery perrow=2 heights=300px widths=300px> File:20170403-qPCR-RCATest-CPLP-ssSynthesis.png|qPCR curves File:2017-04-03-RCATest-CPLP-ssSynthesis-PostBglii.png|Pre-purification gel File:2017-04-03-RCATest-CPLP-ssSynthesis-PostBglii-Purified.png|Post purification gel </gallery> Purification really didn't do much but it the gels look good. The product should be around 200bp, btw, because I only used AmpF, which adds on about 20bp ==Protocol-[[Daniel:Notebook/ComboLock/2017-3-26|Pre-Bglii samples]]== <ol start="2"> <li>Bgl Digestion</li> <ol type="A"> <li>Make the following reactions; '''Do not add Bglii yet'''</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt;font-weight:bold" align="center" |style="background-color:#8DB4E2" width="210" height="42" | Reagent |style="background-color:#8DB4E2" width="85" | Single Rxn Vol |style="background-color:#95B3D7" width="85" | Master Mix (6.2X) |- style="font-size:12pt" | height="15" valign="bottom" | RCA Rxn | align="center" align="center" valign="bottom" | 8 | align="center" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | 10X Buffer 3.1 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 12.4 |- style="font-size:12pt" | height="15" valign="bottom" | Bglii | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 9 | align="center" align="center" valign="bottom" | 55.8 |- style="font-size:12pt" | height="15" valign="bottom" | Total |style="font-weight:bold" align="center" align="center" valign="bottom" | 20 |style="font-weight:bold" align="center" align="center" valign="bottom" | 68.2 |} <li>Use the following thermocycler program</li> <ol type="a"> <li>5 min 95C</li> <li>Ramp to 50C at 0.2C/s</li> <li>10 min 50C</li> <li>1 hr 37C; '''When this cycle starts add 1 uL Bglii'''</li> <li>Heat kill with 65C for 20 min</li></ol> </ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 60 uL 10X TBE and 20 uL 6X dye</li> <li>Aliquot 10 uL on to parafilm per sample, and 10 uL TBE + 2 uL 6X dye to ladder aliquot</li> <li>Add 4 uL reaction or 1.5 uL ladder to appropriate aliquot</li> <li>Add 10 uL mix to gel lanes</li> <li>Run gel for 24 minutes at 230V</li> <li>Stain with 2 uL SYBR gold for 3 minutes</li> <li>Rinse and image in gel doc</li> </ol></ol> [[Image:2017-04-04_-RCATest-CPLP-ssSynthesis-sssThenBgl.png|500px]] It appears that the Bgl digestion was incomplete. That does mean there is a lot more product available than can be cut in an hour with one uL of Bglii, which according to [https://www.neb.com/products/r0144-bglii#pd-protocols NEB] is 10 ug (1 ug/unit/hr) [[Category:ComboLock]] [[Category:20170324]]
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