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=ALU Control= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] I have the ALU primers in and I'm testing whether or not I can amplify working probes from genomic DNA. ==Genomic DNA Extraction== I did this using PGP1F fibroblasts from my cell culture over in CERC. 1. Trypsinize cells using 1 mL Trypsin LE 2. Resuspend in 10 mL DMEM 3. Spin at 1000 rpm for 3 minutes, remove supernatant 4. Resuspend in 1 mL Lysis buffer from Zymo kit 5. Follow Zymo kit instructions for monolayer cells ===Nanodrop Results=== I separated the DNA into two columns, so there are two samples (A and B) {| 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" | width="65" height="15" | Sample | width="65" | PGP1F-A | width="65" | PGP1F-B |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ng/uL | align="center" | 37.4 | align="center" | 27.4 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | ug in 100 uL | align="center" | 3.7 | align="center" | 2.7 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | total | align="center" | | align="center" | 6.5 |} Since I seem to have genomic DNA, I can now test the primers by doing a PCR run. ==qPCR Primary Amplification== This is only a test run, so I'm going to do it in triplicate (if this succeeds I'll have enough DNA to run the DpnII digestion). 1. Reaction table {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" |style="background-color:#FFFF00" align="center" width="65" height="45" | |style="background-color:#D9D9D9;font-weight:bold" width="65" | gDNA |style="background-color:#D9D9D9;font-weight:bold" width="65" | 2X Kapa SYBR qPCR MM |style="background-color:#D9D9D9;font-weight:bold" width="65" | 100 uM AP1Alu |style="background-color:#D9D9D9;font-weight:bold" width="65" | 100 uM AP2Alu |style="background-color:#D9D9D9;font-weight:bold" width="65" | H20 |style="background-color:#D9D9D9;font-weight:bold" width="65" | Total Volume |- style="background-color:#8DB4E2;font-size:12pt" align="center" | height="15" | per rxn | align="center" | 1 | align="center" | 50 | align="center" | 0.5 | align="center" | 0.5 | align="center" | 48 | align="center" | 100 |- style="font-size:12pt" align="center" |style="background-color:#D9D9D9;font-weight:bold" height="30" | PGP1F-A (3.1X) | align="center" | 3.1 | align="center" | 155 | align="center" | 1.55 | align="center" | 1.55 | align="center" | 148.8 | align="center" | 310 |- style="font-size:12pt" align="center" |style="background-color:#D9D9D9;font-weight:bold" height="30" | PGP1F-B (3.1X) | align="center" | 3.1 | align="center" | 155 | align="center" | 1.55 | align="center" | 1.55 | align="center" | 148.8 | align="center" | 310 |} 2. Aliquot 100 uL into each of 3 qPCR tubes 3. PCR protocol: i. PCR 3 min 95C ii. 95C 10 sec iii. 55C 30 sec iv. 72C 20 sec v. plate read vi. goto ii x17 vii. 72C 2 min viii. 16C hold 4. Pool 3 samples together each 5. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB 6. Measure concentrations in nanodrop ===qPCR Results=== [[Image:2013-12-18-ALUTest.png|500px]] ===Nanodrop Results=== {| 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" | width="65" height="15" | Sample | width="65" | PGP1F-A | width="65" | PGP1F-B |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ng/uL | align="center" | 12.9 | align="center" | 13.4 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ug in 40 uL | align="center" | 0.5 | align="center" | 0.5 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | total | align="center" | | align="center" | 1.0 |} So first round PCR is not at all lucrative. This product will still be used for several tests, however. The first test will be for DpnII cleavage. I'll also check to see if second round PCR works. ==DpnII Digestion== 1. Reaction Table {| 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" | width="65" height="15" | Sample | width="65" | PGP1F-A | width="65" | PGP1F-B |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ng/uL | align="center" | 12.9 | align="center" | 13.4 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ug in 30 uL | align="center" | 0.4 | align="center" | 0.4 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | uL 10X buffer | align="center" | 5 | align="center" | 5 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | uL DpnII (50U/uL) | align="center" | 1 | align="center" | 1 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | uL nfH2O | align="center" | 14 | align="center" | 14 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Total | align="center" | 50 | align="center" | 50 |} 2. Incubate samples at 37C for '''2''' hours; 20 minute heat shock at 65C 3. Column Purification, elute with 30 uL EB 4. Measure concentrations in nanodrop ===Nanodrop Results=== {| 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" | width="65" height="15" | Sample | width="65" | PGP1F |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ng/uL | align="center" | 13.4 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ug in 30 uL | align="center" | 0.4 |} ===Gel Image=== [[Image:2013-12-18-ALUTest.jpg|600px]] ==qPCR Secondary Amplification== 1. Reaction Table {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" align="center" |style="background-color:#FFFF00" align="center" width="65" height="45" | |style="background-color:#D9D9D9;font-weight:bold" width="65" | gDNA |style="background-color:#D9D9D9;font-weight:bold" width="65" | 2X Kapa SYBR qPCR MM |style="background-color:#D9D9D9;font-weight:bold" width="65" | 100 uM AP1V61U-Amino |style="background-color:#D9D9D9;font-weight:bold" width="65" | 100 uM AP2V6 |style="background-color:#D9D9D9;font-weight:bold" width="65" | H20 |style="background-color:#D9D9D9;font-weight:bold" width="65" | Total Volume |- style="background-color:#8DB4E2;font-size:12pt" align="center" | height="15" | per rxn | align="center" | 1 | align="center" | 50 | align="center" | 0.5 | align="center" | 0.5 | align="center" | 48 | align="center" | 100 |- style="font-size:12pt" align="center" |style="background-color:#D9D9D9;font-weight:bold" height="30" | PGP1F-A (1X) | align="center" | 1 | align="center" | 50 | align="center" | 0.5 | align="center" | 0.5 | align="center" | 48 | align="center" | 100 |- style="font-size:12pt" align="center" |style="background-color:#D9D9D9;font-weight:bold" height="30" | PGP1F-B (1X) | align="center" | 1 | align="center" | 50 | align="center" | 0.5 | align="center" | 0.5 | align="center" | 48 | align="center" | 100 |} 2. PCR protocol: i. PCR 3 min 95C ii. 95C 10 sec iii. 55C 30 sec iv. 72C 20 sec v. plate read vi. goto ii x17 vii. 72C 2 min viii. 16C hold 3. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 30 uL EB 4. Measure concentrations in nanodrop ===qPCR Results=== [[Image:2013-12-18-ALUTest2.png|500px]] ===Nanodrop Results=== {| 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" | width="65" height="15" | Sample | width="65" | PGP1F-A | width="65" | PGP1F-B |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ng/uL | align="center" | 36.5 | align="center" | 27.4 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ug in 30 uL | align="center" | 1.1 | align="center" | 0.8 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | total | align="center" | | align="center" | 1.9 |}
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