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Daniel:Notebook/HiResChrPaint/2013-7-23
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=S2 Probe Preparation ([[Daniel:Notebook/HiResChrPaint/2013-7-22|Started 7/22/2013]])= [[Daniel:Notebook/HiResChrPaint|Back to Calendar]] ==DpnII Digestion ([[Daniel:Notebook/HiResChrPaint/2013-7-22|from 7/22/2013]])== 3. Pool samples for V4 set into 4AB and 4CD 4. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 30 uL EB** 5. Pooled 4AB and 4CD into V4S2 and pooled V6 sets into V6AB and V6CD 6. Measure concentrations in nanodrop Note (**): Forgot to move the columns to fresh tubes before elution. Caught myself just as the centrifuge started, so I stopped it immediately, before it even got to full speed. Still, this resulted in me losing most of the samples (I also threw away the collection tubes before proceeding to the fresh collection tube). ===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" | V4S2 | width="65" | V6S2AB | width="65" | V6S2CD |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ng/uL | align="center" | 142.9 | align="center" | 32.8 | align="center" | 34.9 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ug in 20 uL | align="center" | 2.9 | align="center" | 0.7 | align="center" | 0.7 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | total ug | align="center" | | align="center" | | align="center" | 1.4 |} ===DpnII Digest gel=== 1. Lanes; (5.5 uL TBE; 2 uL 6x buffer, 0.5 uL sample; load 7 uL for all) {{GelLanes|Lane3=25bp ladder|Lane4=V4S2|Lane5=V6S2AB|Lane6=V4S2CD}} '''Gel Image''' [[Image:DpnIIDigest_72313.jpg|600px]] ===Discussion=== Since I botched this probe prep, I may have to start over. I still have to digest with lambda exonuclease, which is going to drop my concentrations by 50% regardless. This would mean I'd likely have <1 ug for both V4S2 and V6S2 prior to dye coupling. It's just not worth it. I'm going to store these probes, and the next time I make a S2 set I'll add the probes in post DpnII digestion. =Column Loading Trial= I talked with Noi about why we always do an ethanol precipitation after PCR, because we do a column prep anyway. As far as we can tell, the ethanol precipitation condenses the volume so that we don't have to use a lot of columns. I think, however, that we can load the column several times and get the same result. To test this, I'm going to run the same amount of DNA on two different columns. I'll load one once (haploid) and one twice (diploid). For this I'm going to use the AmpF probes that Noi gave me (~50 bp, 1392.5 ng/uL). 1. Add 100 uL nfH2O to hap and 200 uL nfH20 to dip 2. Add 2 uL AmpF to each (~3 ug) 3. Add 600 uL PB to hap and 1200 uL PB (requires 2x spins) 4. Elute with 40 uL PB 5. Measure with Nanodrop ==Nanodrop Results== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" | align="center" width="65" height="15" align="center" | |style="font-weight:bold" width="65" align="center" | ng/uL |style="font-weight:bold" width="65" align="center" | ug in 40 uL |style="font-weight:bold" width="65" valign="bottom" | Yield % |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Haploid | align="center" | 12.8 | align="center" | 0.5 | align="center" | 17.1 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Diploid | align="center" | 11.1 | align="center" | 0.4 | align="center" | 14.8 |} ==Discussion== Although n is only equal to 1, it looks as though multiple loading the column is no problem. This will save on time, as the ethanol precipitation used up at least 4 hours incubation (probably overnight is better) as well as an hour or so centrifugation. I'm going to try skipping the ethanol precipitation post-PCR and going straight to the columns. =S2 Probe Prep= I'm going to retry the S2 probe prep. 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" | | width="65" | 20 nM OligoPool | width="65" | 2X Kapa SYBR qPCR MM | width="65" | 100 uM AP1V41U | width="65" | 100 uM AP2V4 | width="65" | 100 uM AP1V61U | width="65" | 100 uM AP2V6 | width="66" | 2 mM aa-dUTP | width="65" | H20 | width="65" | Total Volume |- style="background-color:#8DB4E2;font-size:12pt" align="center" | height="15" | per rxn | align="center" | 0.2 | align="center" | 50 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 0.4 | align="center" | 2.5 | align="center" | 45.7 | align="center" | 100 |- style="font-size:12pt" align="center" | height="30" | V4S2 (12.5x) | align="center" | 2.5 | align="center" | 625 | align="center" | 5 | align="center" | 5 | align="center" | 0 | align="center" | 0 | align="center" | 31 | align="center" | 581.5 | align="center" | 1250 |- style="font-size:12pt" align="center" | height="30" | V6S2 (12.5x) | align="center" | 2.5 | align="center" | 625 | align="center" | 0 | align="center" | 0 | align="center" | 5 | align="center" | 5 | align="center" | 31 | align="center" | 581.5 | align="center" | 1250 |} 2. Aliquot 100 uL into each of 12 qPCR tubes 3. PCR protocol: i. PCR 3 min 95C ii. 95C 3 sec iii. 55C 30 sec iv. 72C 20 sec v. plate read vi. goto ii x17 vii. 72C 2 min viii. 16C hold 4. Pooled 3 samples together each (300 uL each, 4 pools per set, 8 total) 5. [[Daniel:Protocols/Qiaquick_Column|Column Purification]], elute with 40 uL EB 6. Measure concentrations in nanodrop ===qPCR Results=== [[Image:2013-7-23-S2.png|600px]] ===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" | V4S4A | width="65" | V4S4B | width="65" | V4S4C | width="65" | V4S4D | width="65" | V6S4A | width="65" | V6S4B | width="65" | V6S4C | width="65" | V6S4D |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ng/uL | align="center" | 62.3 | align="center" | 69.7 | align="center" | 69.9 | align="center" | 69.5 | align="center" | 106.6 | align="center" | 109.8 | align="center" | 111.6 | align="center" | 111.4 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ug in 40 uL | align="center" | 2.5 | align="center" | 2.8 | align="center" | 2.8 | align="center" | 2.8 | align="center" | 4.3 | align="center" | 4.4 | align="center" | 4.5 | align="center" | 4.5 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Total ug | align="center" | | align="center" | | align="center" | | align="center" | 10.9 | align="center" | | align="center" | | align="center" | | align="center" | 17.6 |} These results are slightly better from [[Daniel:Notebook/HiResChrPaint/2013-7-22|yesterday's PCR]], but basically the same. So I think the ethanol precipitation step can be skipped. ==DpnII Digest== 1. Prepare Samples {| 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" | V4S4A | width="65" | V4S4B | width="65" | V4S4C | width="65" | V4S4D | width="65" | V6S4A | width="65" | V6S4B | width="65" | V6S4C | width="65" | V6S4D |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ng/uL | align="center" | 52.6 | align="center" | 44.5 | align="center" | 52.1 | align="center" | 47.4 | align="center" | 105.7 | align="center" | 111.3 | align="center" | 98.1 | align="center" | 103.3 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | ug in 35 uL | align="center" | 2.1 | align="center" | 1.8 | align="center" | 2.1 | align="center" | 1.9 | align="center" | 4.2 | align="center" | 4.5 | align="center" | 3.9 | align="center" | 4.1 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | uL 10X buffer | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 | align="center" | 5 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="30" | uL DpnII (50U/uL) | align="center" | 2 | align="center" | 2 | align="center" | 2 | align="center" | 2 | align="center" | 2 | align="center" | 2 | align="center" | 2 | align="center" | 2 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | uL nfH2O | align="center" | 8 | align="center" | 8 | align="center" | 8 | align="center" | 8 | align="center" | 8 | align="center" | 8 | align="center" | 8 | align="center" | 8 |- style="font-size:12pt" align="center" |style="font-weight:bold" height="15" | Total | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 | align="center" | 50 |} 2. Incubate samples at 37C for 16 hours; 20 minute heat shock at 65C; [[Daniel:Notebook/HiResChrPaint/2013-7-24|Continued 7/24/2013]]
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