Daniel:Notebook/HiResChrPaint/2013-6-5: Difference between revisions
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>Djacobse (Created page with "=Probe Prep 3 (Started 5/29/2013)= Back to Calendar ==DpnII Digestion== Finishing the digest ...") |
>Djacobse No edit summary |
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(4 intermediate revisions by 2 users not shown) | |||
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1. Set up | 1. Set up | ||
C group | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| 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" valign="bottom" | |- style="font-size:12pt;font-weight:bold" align="center" valign="bottom" | ||
Line 20: | Line 21: | ||
|- style="font-size:12pt" align="center" valign="bottom" | |- style="font-size:12pt" align="center" valign="bottom" | ||
| height="15" | alpha 4- | | height="15" | alpha 4-C | ||
| align="center" | 35 | | align="center" | 35 | ||
| align="center" | 2 | | align="center" | 2 | ||
Line 28: | Line 29: | ||
|- style="font-size:12pt" align="center" valign="bottom" | |- style="font-size:12pt" align="center" valign="bottom" | ||
| height="15" | alpha 6- | | height="15" | alpha 6-C | ||
| align="center" | 35 | | align="center" | 35 | ||
| align="center" | 2 | | align="center" | 2 | ||
Line 36: | Line 37: | ||
|- style="font-size:12pt" align="center" valign="bottom" | |- style="font-size:12pt" align="center" valign="bottom" | ||
| height="15" | beta 4- | | height="15" | beta 4-C | ||
| align="center" | 35 | | align="center" | 35 | ||
| align="center" | 2 | | align="center" | 2 | ||
Line 44: | Line 45: | ||
|- style="font-size:12pt" align="center" valign="bottom" | |- style="font-size:12pt" align="center" valign="bottom" | ||
| height="15" | beta 6- | | height="15" | beta 6-C | ||
| align="center" | 35 | |||
| align="center" | 2 | |||
| align="center" | 5 | |||
| align="center" | 8 | |||
| align="center" | 50 | |||
|} | |||
D Group | |||
{| 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" valign="bottom" | |||
| width="65" height="30" | Sample | |||
| width="65" | uL sample | |||
| width="65" | uL Dpn II (50 U/uL) | |||
| width="65" | uL 10x buffer | |||
| width="65" | uL H20 | |||
| width="65" | total | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | alpha 4-D | |||
| align="center" | 35 | |||
| align="center" | 2 | |||
| align="center" | 5 | |||
| align="center" | 8 | |||
| align="center" | 50 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | alpha 6-D | |||
| align="center" | 35 | |||
| align="center" | 2 | |||
| align="center" | 5 | |||
| align="center" | 8 | |||
| align="center" | 50 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | beta 4-D | |||
| align="center" | 35 | |||
| align="center" | 2 | |||
| align="center" | 5 | |||
| align="center" | 8 | |||
| align="center" | 50 | |||
|- style="font-size:12pt" align="center" valign="bottom" | |||
| height="15" | beta 6-D | |||
| align="center" | 35 | | align="center" | 35 | ||
| align="center" | 2 | | align="center" | 2 | ||
Line 54: | Line 99: | ||
2. Incubate in thermocycler at 37C for 16 hours; heat shock 20 minutes at 65 C | 2. Incubate in thermocycler at 37C for 16 hours; heat shock 20 minutes at 65 C | ||
3. [[Daniel:Protocols/Qiaquick_Column|Column purification]], elute with 30 uL EB | |||
4. Measure 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" valign="bottom" | |||
| width="65" height="15" | Sample | |||
| width="65" | a-4C | |||
| width="65" | a-6C | |||
| width="65" | b-4C | |||
| width="65" | b-6C | |||
| width="65" | a-4D | |||
| width="65" | a-6D | |||
| width="65" | b-4D | |||
| width="65" | b-6D | |||
|- style="font-size:12pt" valign="bottom" | |||
|style="font-weight:bold" height="15" | ng/uL | |||
| align="center" | 119.1 | |||
| align="center" | 119.2 | |||
| align="center" | 108.5 | |||
| align="center" | 140.4 | |||
| align="center" | 121.8 | |||
| align="center" | 147.7 | |||
| align="center" | 112.2 | |||
| align="center" | 128.7 | |||
|- style="font-size:12pt" valign="bottom" | |||
|style="font-weight:bold" height="15" | ug in 30 uL | |||
| align="center" | 3.6 | |||
| align="center" | 3.6 | |||
| align="center" | 3.3 | |||
| align="center" | 4.2 | |||
| align="center" | 3.7 | |||
| align="center" | 4.4 | |||
| align="center" | 3.4 | |||
| align="center" | 3.9 | |||
|- style="font-size:12pt" valign="bottom" | |||
|style="font-weight:bold" height="15" | Yield (%) | |||
| align="center" | 78.0 | |||
| align="center" | 73.7 | |||
| align="center" | 80.1 | |||
| align="center" | 85.2 | |||
| align="center" | 80.1 | |||
| align="center" | 81.6 | |||
| align="center" | 77.0 | |||
| align="center" | 77.1 | |||
|} | |||
===TBE Gel=== | |||
Check to make sure samples are at least mostly digested. | |||
1. Setup | |||
{| 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" | Lane | |||
| align="center" width="50" | 1 | |||
| align="center" width="50" | 2 | |||
| align="center" width="50" | 3 | |||
| align="center" width="50" | 4 | |||
| align="center" width="50" | 5 | |||
| align="center" width="67" | 6 | |||
| align="center" width="50" | 7 | |||
| align="center" width="50" | 8 | |||
| align="center" width="50" | 9 | |||
| align="center" width="50" | 10 | |||
|- style="font-size:12pt" align="center" | |||
|style="font-weight:bold" height="30" | Sample | |||
| 25 bp ladder | |||
| V4S2C | |||
| V6S2C | |||
| V4S2C* | |||
| V6S2C* | |||
| Undigested | |||
| V4S2D | |||
| V6S2D | |||
| V4S2D* | |||
| V6S2D* | |||
|} | |||
Note: *denotes samples without aa-dUTP | |||
2. Ran gel for 25 minutes at 220 V | |||
3. Stained for 1 minute with 3 uL SYBR gold | |||
[[Image:DpnDigest_060513.jpg|600px]] |
Latest revision as of 23:59, 5 June 2013
Probe Prep 3 (Started 5/29/2013)[edit]
DpnII Digestion[edit]
Finishing the digest on the PCR amplified probes, aa-dUTP and no aa-dUTP. Will run for 2 hours since I didn't see any qualitative difference between the two.
1. Set up
C group
Sample | uL sample | uL Dpn II (50 U/uL) | uL 10x buffer | uL H20 | total |
alpha 4-C | 35 | 2 | 5 | 8 | 50 |
alpha 6-C | 35 | 2 | 5 | 8 | 50 |
beta 4-C | 35 | 2 | 5 | 8 | 50 |
beta 6-C | 35 | 2 | 5 | 8 | 50 |
D Group
Sample | uL sample | uL Dpn II (50 U/uL) | uL 10x buffer | uL H20 | total |
alpha 4-D | 35 | 2 | 5 | 8 | 50 |
alpha 6-D | 35 | 2 | 5 | 8 | 50 |
beta 4-D | 35 | 2 | 5 | 8 | 50 |
beta 6-D | 35 | 2 | 5 | 8 | 50 |
2. Incubate in thermocycler at 37C for 16 hours; heat shock 20 minutes at 65 C 3. Column purification, elute with 30 uL EB 4. Measure in Nanodrop
Nanodrop Results[edit]
Sample | a-4C | a-6C | b-4C | b-6C | a-4D | a-6D | b-4D | b-6D |
ng/uL | 119.1 | 119.2 | 108.5 | 140.4 | 121.8 | 147.7 | 112.2 | 128.7 |
ug in 30 uL | 3.6 | 3.6 | 3.3 | 4.2 | 3.7 | 4.4 | 3.4 | 3.9 |
Yield (%) | 78.0 | 73.7 | 80.1 | 85.2 | 80.1 | 81.6 | 77.0 | 77.1 |
TBE Gel[edit]
Check to make sure samples are at least mostly digested.
1. Setup
Lane | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
Sample | 25 bp ladder | V4S2C | V6S2C | V4S2C* | V6S2C* | Undigested | V4S2D | V6S2D | V4S2D* | V6S2D* |
Note: *denotes samples without aa-dUTP
2. Ran gel for 25 minutes at 220 V 3. Stained for 1 minute with 3 uL SYBR gold