Daniel:Notebook/HiResChrPaint/2013-4-29: Difference between revisions
Jump to navigation
Jump to search
>Djacobse (Created page with "=Probe Prep Test (Started 4/22/2013)= Back to calendar =TBE Gel= Purpose: As per [[Daniel:Not...") |
>Djacobse No edit summary |
||
(4 intermediate revisions by the same user not shown) | |||
Line 32: | Line 32: | ||
|} | |} | ||
2. Ran gel for | 2. Ran gel for 25 minutes at 200 V | ||
3. Stained with 3 uL SYBR gold for 5 min | 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]] |
Latest revision as of 23:17, 29 April 2013
Probe Prep Test (Started 4/22/2013)[edit]
TBE Gel[edit]
Purpose: As per 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[edit]
1. Prepared samples (1 uL sample, 5 uL TBE, 2 uL 6x dye) and loaded (7 uL) according to table
Lane | 2 | 3 | 4 | 5 | 6 |
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[edit]
Lambda Exonuclease Digestion[edit]
1. Set up following reaction *Lambda Exo: NEB M0262L; Buffer: NEB B0262S
Sample | ng/uL | uL | ng | Incubation Time | uL buffer | uL lambda exo (5U/uL) | uL nf H20 | Total |
V6S2 | 27.3 | 25 | 682.5 | 1 hr | 5 | 0.5 | 19.5 | 50 |
V6S3 | 27.6 | 25 | 690 | 1 hr | 5 | 2 | 18 | 50 |
V6S4 | 25.3 | 25 | 632.5 | 30 min | 5 | 2 | 18 | 50 |
V4S2 | 37 | 25 | 925 | 2 hr | 5 | 2 | 18 | 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[edit]
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 4/30/2013