Daniel:Notebook/HiResChrPaint/2013-6-5: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Djacobse
(Created page with "=Probe Prep 3 (Started 5/29/2013)= Back to Calendar ==DpnII Digestion== Finishing the digest ...")
 
>Djacobse
No edit summary
 
(4 intermediate revisions by 2 users not shown)
Line 10: Line 10:
  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-B
| 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-B
| 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-B
| 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-B
| 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]

Back to Calendar

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

File:DpnDigest 060513.jpg