Haynes Lab:Notebook/Engineering PC-TFs/2014/06/09

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(Autocreate 2014/06/09 Entry for Haynes_Lab:Notebook/Engineering_PC-TFs)
Current revision (15:15, 24 June 2014) (view source)
(Summary)
 
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==Summary==
==Summary==
-
*
+
'''Digest All the Things'''<br>
 +
----
 +
'''Well One: Ladder'''<br>
 +
'''Well Two'''<br>
 +
{| {{table}} border="1" cellspacing="3"
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | Reagent
 +
| bgcolor=#cfcfcf | Volume
 +
| rowspan="7" | <u>Expected:</u><br>1. Unknown <br><u>Observed:</u><br> 3k, 7k, 2k<br>
 +
|-
 +
| DNA(CMV/MV9) || 5.0 μL
 +
|-
 +
| 10X buffer || 1.5 μL
 +
|-
 +
| XbaI || 0.0 μL
 +
|-
 +
| NruI || 0.0 μL
 +
|-
 +
| dH<sub>2</sub>O || 8.5 μL
 +
|-
 +
| Total || 15 μL --> 37°C/ 30 min.
 +
|}
 +
<br>
 +
'''Well Three'''<br>
 +
{| {{table}} border="1" cellspacing="3"
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | Reagent
 +
| bgcolor=#cfcfcf | Volume
 +
| rowspan="7" | <u>Expected:</u><br>1. 5200bp <br><u>Observed:</u><br> 5.2k, 2k<br>
 +
|-
 +
| DNA(CMV/MV9) || 5.0 μL
 +
|-
 +
| 10X buffer || 1.5 μL
 +
|-
 +
| XbaI || 1.0 μL
 +
|-
 +
| NruI || 0.0 μL
 +
|-
 +
| dH<sub>2</sub>O || 7.5 μL
 +
|-
 +
| Total || 15 μL --> 37°C/ 30 min.
 +
|}<br>
 +
'''Well Four'''<br>
 +
{| {{table}} border="1" cellspacing="3"
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | Reagent
 +
| bgcolor=#cfcfcf | Volume
 +
| rowspan="7" | <u>Expected:</u><br>1. 5200bp <br><u>Observed:</u><br> 5.2k, 2k<br>
 +
|-
 +
| DNA(CMV/MV9) || 5.0 μL
 +
|-
 +
| 10X buffer || 1.5 μL
 +
|-
 +
| XbaI || 0.0 μL
 +
|-
 +
| NruI || 1.0 μL
 +
|-
 +
| dH<sub>2</sub>O || 7.5 μL
 +
|-
 +
| Total || 15 μL --> 37°C/ 30 min.
 +
|}
 +
<br>
 +
'''Well Five'''<br>
 +
{| {{table}} border="1" cellspacing="3"
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | Reagent
 +
| bgcolor=#cfcfcf | Volume
 +
| rowspan="7" | <u>Expected:</u><br>1. 710bp, 4490bp <br><u>Observed:</u><br> 4k, 2k, 700<br>
 +
|-
 +
| DNA(CMV/MV9) || 5.0 μL
 +
|-
 +
| 10X buffer || 1.5 μL
 +
|-
 +
| XbaI || 1.0 μL
 +
|-
 +
| NruI || 1.0 μL
 +
|-
 +
| dH<sub>2</sub>O || 6.5 μL
 +
|-
 +
| Total || 15 μL --> 37°C/ 30 min.
 +
|}
 +
<br>'''Well Six'''<br>
 +
{| {{table}} border="1" cellspacing="3"
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | Reagent
 +
| bgcolor=#cfcfcf | Volume
 +
| rowspan="7" | <u>Expected:</u><br>1. BL01 = Insert (2520bp), V0120(3265bp) <br><u>Observed:</u><br> 3.1k, 2.6k<br>
 +
|-
 +
| DNA(BL01A) || 5.0 μL
 +
|-
 +
| 10X buffer || 1.5 μL
 +
|-
 +
| XbaI || 1.0 μL
 +
|-
 +
| SpeI || 1.0 μL
 +
|-
 +
| dH<sub>2</sub>O || 7.5 μL
 +
|-
 +
| Total || 15 μL --> 37°C/ 30 min.
 +
|}
 +
<br>'''Well Seven'''<br>
 +
{| {{table}} border="1" cellspacing="3"
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | Reagent
 +
| bgcolor=#cfcfcf | Volume
 +
| rowspan="7" | <u>Expected:</u><br>1. BL01 = 2520bp, 3265bp <br> <u>Observed:</u><br> 3.1k, 2.6k, 7k<br>
 +
|-
 +
| DNA(BL01B) || 5.0 μL
 +
|-
 +
| 10X buffer || 1.5 μL
 +
|-
 +
| XbaI || 1.0 μL
 +
|-
 +
| SpeI || 1.0 μL
 +
|-
 +
| dH<sub>2</sub>O || 7.5 μL
 +
|-
 +
| Total || 15 μL --> 37°C/ 30 min.
 +
|}
 +
<br>'''Well Eight'''<br>
 +
{| {{table}} border="1" cellspacing="3"
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | Reagent
 +
| bgcolor=#cfcfcf | Volume
 +
| rowspan="7" | <u>Expected:</u><br> 2520bp, 3265bp <u>Observed:</u><br> Faint: 3.1k, 2.6k, 7k<br>
 +
|-
 +
| DNA(BL05) || 5.0 μL
 +
|-
 +
| 10X buffer || 1.5 μL
 +
|-
 +
| XbaI || 1.0 μL
 +
|-
 +
| SpeI || 1.0 μL
 +
|-
 +
| dH<sub>2</sub>O || 7.5 μL
 +
|-
 +
| Total || 15 μL --> 37°C/ 30 min.
 +
|}
 +
<br>'''Well Nine'''<br>
 +
{| {{table}} border="1" cellspacing="3"
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | Reagent
 +
| bgcolor=#cfcfcf | Volume
 +
| rowspan="7" | <u>Expected:</u><br> 1431bp, 3265bp <u>Observed:</u><br> 3.1k, 4.9k, 1.5k <br>
 +
|-
 +
| DNA(BL09) || 5.0 μL
 +
|-
 +
| 10X buffer || 1.5 μL
 +
|-
 +
| XbaI || 1.0 μL
 +
|-
 +
| SpeI || 1.0 μL
 +
|-
 +
| dH<sub>2</sub>O || 7.5 μL
 +
|-
 +
| Total || 15 μL --> 37°C/ 30 min.
 +
|}
 +
<br>'''Well 10'''<br>
 +
{| {{table}} border="1" cellspacing="3"
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | Reagent
 +
| bgcolor=#cfcfcf | Volume
 +
| rowspan="7" | <u>Expected:</u><br>1. BL01 = 7720bp <br> <u>Observed:</u><br> 4k, 2k, 1.5k <br>
 +
|-
 +
| DNA(CMV/MV9 & BL01) || 5.0 μL
 +
|-
 +
| 10X buffer || 1.5 μL
 +
|-
 +
| XbaI || 0.0 μL
 +
|-
 +
| NruI || 1.0 μL
 +
|-
 +
| dH<sub>2</sub>O || 7.5 μL
 +
|-
 +
| Total || 15 μL --> 37°C/ 30 min.
 +
|}
 +
<br>'''Well 11'''<br>
 +
{| {{table}} border="1" cellspacing="3"
 +
|- valign="top"
 +
| bgcolor=#cfcfcf | Reagent
 +
| bgcolor=#cfcfcf | Volume
 +
| rowspan="7" | <u>Expected:</u><br>1. dependent on ligation direction <br><u>Observed:</u><br>4k, 2k, 1.5k<br>
 +
|-
 +
| DNA(CMV/MV9 & BL01) || 5.0 μL
 +
|-
 +
| 10X buffer || 1.5 μL
 +
|-
 +
| XbaI || 1.0 μL
 +
|-
 +
| NruI || 1.0 μL
 +
|-
 +
| dH<sub>2</sub>O || 6.5 μL
 +
|-
 +
| Total || 15 μL --> 37°C/ 30 min.
 +
|}
 +
<br>
 +
----
 +
'''Gel Verification'''
 +
{| {{table}} border="1" cellspacing="3" <!-- Plates. table -->
 +
|- valign="top"
 +
| rowspan="7" | [[Image:20140609 172345.jpg|400px|Hover name]] <br> [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
 +
|}
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

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Summary

Digest All the Things


Well One: Ladder
Well Two

Reagent Volume Expected:
1. Unknown
Observed:
3k, 7k, 2k
DNA(CMV/MV9) 5.0 μL
10X buffer 1.5 μL
XbaI 0.0 μL
NruI 0.0 μL
dH2O 8.5 μL
Total 15 μL --> 37°C/ 30 min.


Well Three

Reagent Volume Expected:
1. 5200bp
Observed:
5.2k, 2k
DNA(CMV/MV9) 5.0 μL
10X buffer 1.5 μL
XbaI 1.0 μL
NruI 0.0 μL
dH2O 7.5 μL
Total 15 μL --> 37°C/ 30 min.

Well Four

Reagent Volume Expected:
1. 5200bp
Observed:
5.2k, 2k
DNA(CMV/MV9) 5.0 μL
10X buffer 1.5 μL
XbaI 0.0 μL
NruI 1.0 μL
dH2O 7.5 μL
Total 15 μL --> 37°C/ 30 min.


Well Five

Reagent Volume Expected:
1. 710bp, 4490bp
Observed:
4k, 2k, 700
DNA(CMV/MV9) 5.0 μL
10X buffer 1.5 μL
XbaI 1.0 μL
NruI 1.0 μL
dH2O 6.5 μL
Total 15 μL --> 37°C/ 30 min.


Well Six

Reagent Volume Expected:
1. BL01 = Insert (2520bp), V0120(3265bp)
Observed:
3.1k, 2.6k
DNA(BL01A) 5.0 μL
10X buffer 1.5 μL
XbaI 1.0 μL
SpeI 1.0 μL
dH2O 7.5 μL
Total 15 μL --> 37°C/ 30 min.


Well Seven

Reagent Volume Expected:
1. BL01 = 2520bp, 3265bp
Observed:
3.1k, 2.6k, 7k
DNA(BL01B) 5.0 μL
10X buffer 1.5 μL
XbaI 1.0 μL
SpeI 1.0 μL
dH2O 7.5 μL
Total 15 μL --> 37°C/ 30 min.


Well Eight

Reagent Volume Expected:
2520bp, 3265bp Observed:
Faint: 3.1k, 2.6k, 7k
DNA(BL05) 5.0 μL
10X buffer 1.5 μL
XbaI 1.0 μL
SpeI 1.0 μL
dH2O 7.5 μL
Total 15 μL --> 37°C/ 30 min.


Well Nine

Reagent Volume Expected:
1431bp, 3265bp Observed:
3.1k, 4.9k, 1.5k
DNA(BL09) 5.0 μL
10X buffer 1.5 μL
XbaI 1.0 μL
SpeI 1.0 μL
dH2O 7.5 μL
Total 15 μL --> 37°C/ 30 min.


Well 10

Reagent Volume Expected:
1. BL01 = 7720bp
Observed:
4k, 2k, 1.5k
DNA(CMV/MV9 & BL01) 5.0 μL
10X buffer 1.5 μL
XbaI 0.0 μL
NruI 1.0 μL
dH2O 7.5 μL
Total 15 μL --> 37°C/ 30 min.


Well 11

Reagent Volume Expected:
1. dependent on ligation direction
Observed:
4k, 2k, 1.5k
DNA(CMV/MV9 & BL01) 5.0 μL
10X buffer 1.5 μL
XbaI 1.0 μL
NruI 1.0 μL
dH2O 6.5 μL
Total 15 μL --> 37°C/ 30 min.



Gel Verification

Hover name
Ladder


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