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=Alu Control= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] Following the [[Daniel:Notebook/HiResChrPaint/2013-12-18|results from December 18]], it appears that my primers work. However, I used up all the sample from the previous primary amplification, so I'll have to re-amplify from genomic DNA ([[Daniel:Notebook/HiResChrPaint/2013-12-18|see Genomic DNA Extraction Results]]). ==Expansion qPCR== 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 (12.1X) | align="center" | 12.1 | align="center" | 605 | align="center" | 6.05 | align="center" | 6.05 | align="center" | 580.8 | align="center" | 1210 |} 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:2014-01-08-Alu1.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" | ALU1-A | width="65" | ALU1-B | width="65" | ALU1-C | width="65" | ALU1-D |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ng/uL | align="center" | 15.4 | align="center" | 13.5 | align="center" | 15 | align="center" | 15.9 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ug in 40 uL | align="center" | 0.6 | align="center" | 0.5 | align="center" | 0.6 | align="center" | 0.6 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | total | align="center" | | align="center" | | align="center" | | align="center" | 2.4 |} ===qPCR Gel=== 1. '''For Each Sample''': 1 uL sample, 5 uL TBE, 2 uL dye; load 7.2 uL {{GelLanes|Lane2=10bp ladder|Lane3=Alu1-A|Lane4=Alu1-B|Lane5=Alu1-C|Lane6=Alu1-D}} 2. Run gel for 30 minutes at 220V 3. Stain 2 minutes with 3 uL SYBR gold '''Gel Image''' [[Image:2014-01-08-Alu1.jpg|500px]] So these appear to all bee the same, and pretty similar to the gel image from [[Daniel:Notebook/HiResChrPaint/2013-12-18|12/18]]. So I'm going to pool the samples and proceed with production qPCR. Pooled sample concentration: '''17.1 ng/uL'''. ==Production qPCR== 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 AP1V6-Am |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 (12.1X) | align="center" | 12.1 | align="center" | 605 | align="center" | 6.05 | align="center" | 6.05 | align="center" | 580.8 | align="center" | 1210 |} 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:2014-01-08Alu2.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" | ALU2-A | width="65" | ALU2-B | width="65" | ALU2-C | width="65" | ALU2-D |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ng/uL | align="center" | 25.1 | align="center" | 25 | align="center" | 25 | align="center" | 22.2 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ug in 40 uL | align="center" | 1.0 | align="center" | 1.0 | align="center" | 1.0 | align="center" | 0.9 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | total | align="center" | | align="center" | | align="center" | | align="center" | 3.9 |} So this is smaller than my usual input material. One possibility is the number of cycles. I could only run this one for 17 cycles, which with the probes is more than enough, but from the [[Daniel:Notebook/HiResChrPaint/2013-12-18|12/18 results]] I'd say that the secondary amplification should be run 21 cycles or so.
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