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Daniel:Notebook/HiResChrPaint/2013-4-29
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=Probe Prep Test ([[Daniel:Notebook/HiResChrPaint/2013-4-22|Started 4/22/2013]])= [[Daniel:Notebook/HiResChrPaint|Back to calendar]] =TBE Gel= Purpose: As per [[Daniel:Notebook/HiResChrPaint/2013-4-27|4/27/2013]] results, I need to better understand the lambda exonuclease digestion. To this end I'm going to play around with the parameters of the reaction. To do so I need several samples to work with. I am testing/identifying samples right now that will suit my purposes. ==Protocol== 1. Prepared samples (1 uL sample, 5 uL TBE, 2 uL 6x dye) and loaded (7 uL) according to table {| 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" | Lane | align="center" width="65" | 2 | align="center" width="65" | 3 | align="center" width="65" | 4 | align="center" width="65" | 5 | align="center" width="65" | 6 |- style="font-size:12pt" align="center" | height="15" | Sample | Ladder | V4S2 | V6S2 | V6S3 | V6S3 Digest |} 2. Ran gel for 25 minutes at 200 V 3. Stained with 3 uL SYBR gold for 5 min ===Gel Results=== [[Image:TBE_042913.jpg|600px]] ==Lambda Exonuclease Digestion== [[Daniel:Reagents/LambdaExonuclease| Enzyme Details]] 1. Set up following reaction *Lambda Exo: NEB M0262L; Buffer: NEB B0262S {| 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="30" | Sample | width="65" | ng/uL | width="65" | uL | width="65" | ng | width="59" | Incubation Time | width="65" | uL buffer | width="65" | uL lambda exo (5U/uL) | width="65" | uL nf H20 | width="65" | Total |- style="font-size:12pt" align="center" | height="15" | V6S2 | align="center" | 27.3 | align="center" | 25 | align="center" | 682.5 | 1 hr | align="center" | 5 | align="center" | 0.5 | align="center" | 19.5 | align="center" | 50 |- style="font-size:12pt" align="center" | height="15" | V6S3 | align="center" | 27.6 | align="center" | 25 | align="center" | 690 | 1 hr | align="center" | 5 | align="center" | 2 | align="center" | 18 | align="center" | 50 |- style="font-size:12pt" align="center" | height="15" | V6S4 | align="center" | 25.3 | align="center" | 25 | align="center" | 632.5 | 30 min | align="center" | 5 | align="center" | 2 | align="center" | 18 | align="center" | 50 |- style="font-size:12pt" align="center" | height="15" | V4S2 | align="center" | 37 | align="center" | 25 | align="center" | 925 | 2 hr | align="center" | 5 | align="center" | 2 | align="center" | 18 | align="center" | 50 |} 3. Incubated in 37C incubator; heat shocked for 20 minutes at 65 C in thermocycler 4. Immediately proceeded to EtOH precipitation with every sample ==Ethanol Precipitation== [[Daniel:Protocols/EtOHPrecip| Standard Protocol]] 1. Added 250 uL nuclease free water to each reaction 2. Added 900 uL 100% EtOH, 30 uL NaOAc, 1 uL glycoblue 3. Incubate overnight at -80C 4. Continued [[Daniel:Notebook/HiResChrPaint/2013-4-30|4/30/2013]]
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