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==Bglii Digest (Sense)== From the results [[Daniel:Notebook/ComboLock/2017-3-3|last week]], I realized that the original primer for Bglii cutting (the DNA must be double stranded and the RCA product is single stranded) was in the wrong sense. This is because the RCA product will be the reverse complement of the circular product. So I'm repeating the Bglii digest from [[Daniel:Notebook/ComboLock/2017-3-2|last Thursday]] using the correct primer. Also, because I'm having trouble keeping track of everything, here are all the letter codes used for the experiment. For future readers, just pay attention to the conditions and maybe sample numbers (Sample 1: Sample, 2: No Template, 3: No C Probe, 4: No PO4 on padlock). '''Sample Matrix''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#9BBB59;font-size:12pt;font-weight:bold" align="center" | width="80" height="40" | Letter Code | width="240" | Condition | width="80" | Samples |- style="font-size:12pt" align="center" valign="bottom" | height="15" | X | PostRCA-Unpurified | 1-4 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" | height="15" | Y | PostRCA-Purified | 1-4 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | Z | Unpurified post Bglii Digest (X) | 1, 4 |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" | height="15" | Q | Purified post Bglii Digest (Y) | 1, 4 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | S | MaxRCA-Unpurified | 1-4 |- style="background-color:#DA9694;font-size:12pt" align="center" valign="bottom" | height="15" | T | MaxRCA-Purified | 1-4 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | V | Unpurified post Bglii Digest (S) | 1, 4 |- style="background-color:#DA9694;font-size:12pt" align="center" valign="bottom" | height="15" | W | Purified post Bglii Digest (T) | 1, 4 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | J | Unpurified Bglii Digest-Sense (X) | 1-4 |- style="background-color:#B1A0C7;font-size:12pt" align="center" valign="bottom" | height="15" | K | Purified Bglii Digest-Sense (Y) | 1-4 |} <ol> <li>BglII Digestion</li> <ol type="A"> <li>Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest</li> <li>Make the following 16.2X master mix</li> {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center" | width="190" height="30" | Reagent | width="85" | Single Rxn Vol (uL) | width="85" | 16.2X MM uL |- style="font-size:12pt" | height="15" valign="bottom" | RCA Reaction Product | align="center" align="center" valign="bottom" | 5 | align="center" align="center" valign="bottom" | 0 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Bglii Cutter Primer (10 uM) | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 32.4 |- style="font-size:12pt" | height="15" valign="bottom" | 10X Buffer 3.1 | align="center" align="center" valign="bottom" | 2 | align="center" align="center" valign="bottom" | 32.4 |- style="background-color:#BFBFBF;font-size:12pt" | height="15" valign="bottom" | Bglii | align="center" align="center" valign="bottom" | 1 | align="center" align="center" valign="bottom" | 0 |- style="font-size:12pt" | height="15" valign="bottom" | nfH2O | align="center" align="center" valign="bottom" | 10 | align="center" align="center" valign="bottom" | 162 |- style="background-color:#BFBFBF;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" | 226.8 |} <li>Aliquot 14 uL master mix into new tubes</li> <li>Add 5 uL appropriate sample to each tube</li> <li>Use the following thermocycler program</li> <ol type="a"> <li>5 min 95C</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>qPCR</li> <ol type="A"> <li>Make 17.2X qPCR master mix according to following recipe</li> <ol type="a"> <li>361.2 uL nfH2O</li> <li>430 uL SYBR Fast</li> <li>17.2 uL 10 uM AmpF</li> <li>17.2 uL 10 uM AmpR-Ind21</li> </ol> <li>Add 48 uL master mix to each well</li> <li>Add 2 uL sample according to plate layout</li> [[Image:PlateLayout-20170308-Padlock0601-BgliiSense.png|600px]] <li>qPCR Cycles</li> <ol type="a"> <li>95C 3 min</li> <li>95C 3 sec</li> <li>55C 30 sec</li> <li>72C 20 sec</li> <li>plate read</li> <li>goto b x30</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> <li>TBE Gel</li> <ol type="A"> <li>Mix 176 uL TBE, 44 uL 6x loading dye</li> <li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> <li>Add 2 uL of sample or ladder to correct drop</li> <li>Load 10 uL in to well</li> <li>Run gel for 23 minutes at 230V</li> <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> <li>Rinse gel and image in gel doc</li> </ol> ===Results=== <gallery perrow=2 heights=300px widths=300px> File:20170308-qPCR-Padlock0601-Bglii-Sense.png|qPCR curves File:2017-03-08-Padlock0601-BgliiSense-Unpurified.png|Gel image - unpurified samples File:2017-03-08-Padlock0601-BgliiSense-Purified.png|Gel image - purified samples </gallery>
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