Team:Freiburg/Notebook/19 July

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<a href="https://2011.igem.org/Team:Freiburg/Notebook"> 19 July </a>
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==<span style="color:green;">green light receptor</span>==
==<span style="color:green;">green light receptor</span>==
-
===NAME OF YOUR EXPERIMENT===
+
===Testdigest with PvuI===
-
'''Investigators:NAME'''
+
'''Investigators:Julia'''
 +
<br/>
 +
digested were Minipreps of Ligationreaction with CcaS.<br/> The Enzym PvuI should cut into two fragments, one of 1666bp, the other 582 bp and vector at 2kb.<br/>
 +
Protocol:<br/>
 +
<br/>
 +
for Mastermix of 7 reactions:<br/>
 +
<br/>
 +
30µl H2O<br/>
 +
7µl NEB buffer 3<br/>
 +
7µl BSA 10x<br/>
 +
2µl PvuI<br/>
 +
<br/>
 +
give 3µl of DNA sample to 7µl Mastermix, digest for one hour.<br/>
 +
Add 1µl loading dye buffer(6x)a nd let the gel run.
 +
====Gelpicture====
==<span style="color:blue;">blue light receptor</span>==
==<span style="color:blue;">blue light receptor</span>==
Line 13: Line 42:
'''Investigators: Sandra, Sophie'''
'''Investigators: Sandra, Sophie'''
-
Primer Degin for Blue light sensor (lovTAP) + trp promotor and tetR Gen + tetO promotor<br/>
+
Primer Degin for Blue light sensor (lovTAP) + trp promotor (BBa_K322999) and tetR Gen + tetO promotor (BBa_Q04400).<br/>
<br/>
<br/>
-
LOVtap-Trp_up gaattcgcggccgcttctagtcacacaggaaagtactatgt<br/>
+
*LOVtap-Trp_up: gaattcgcggccgcttctagtcacacaggaaagtactatgt<br/>
-
LOVtap-Trp_dw tacttttatctaatctggacatctagtatttctcctctttgtcgataccctttttacgtg<br/>
+
*LOVtap-Trp_dw: tacttttatctaatctggacatctagtatttctcctctttgtcgataccctttttacgtg<br/>
-
TetR-TetO_up aaagaggagaaatactagatgtccagattag<br/>
+
*TetR-TetO_up: aaagaggagaaatactagatgtccagattag<br/>
-
TetR-TetO_dw* ctgcagcggccgctactag<br/>
+
*TetR-TetO_dw^: ctgcagcggccgctactag<br/>
-
*we also have another primer for this one, but we are not sure about the length/melting temperature. Might be changed.<br/>
+
^we also have another primer for this one, but we are not sure about the length/melting temperature. Might be changed.<br/>
==<span style="color:red;">red light receptor</span>==
==<span style="color:red;">red light receptor</span>==
-
===NAME OF YOUR EXPERIMENT===
+
===Testdigest===
-
'''Investigators:NAME'''
+
'''Investigators: Jakob'''
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name: Jakob
 +
 +
 +
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 19.07.2011
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Experiment :
 +
 +
16.07.2011
 +
 +
Miniprep
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: Red light receptor
 +
 +
|}
 +
For one reaction you need: For Mastermix: Number of samples+2extra
 +
 +
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 4μl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| H<sub>2</sub>O
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 24
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1μl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Buffer, NEB4
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 6
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1μl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| BSA (10x)
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 6
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 0,5 μl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Enzym 1
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 0,5 μl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Enzym 2
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 3 μl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 3
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 +
|}
 +
10 μl total volume
 +
 +
 +
Give 3 μl of DNA in an eppi and add 7μl of the mastermix.
 +
 +
Incubate for about 1h at 37°C.
 +
 +
 +
Add 1 μl Loading dye buffer and load the gel.
 +
 +
Picture of the tesdigest: Expected sizes: L22+term(L21) ≈ 800 bp, L23+term(L21) ≈ 800 bp
 +
[[File:Freiburg 11 19 07 2011 testdigest jakob L22a-L23b.JPG|800px]]
 +
 +
L22a+b are correct
 +
 +
*To-do: send for sequencing
 +
 +
===Miniprep===
 +
 +
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name:
 +
 +
Jakob
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date:
 +
 +
19.07.2011
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Experiment: 18.07.2011
 +
 +
Trafo and Grow overnight at 37 °C
 +
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name:
 +
 +
Red light receptor
 +
 +
|}
 +
'''Documentation:'''
 +
 +
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Part name: L22+L21 = ho1 + terminator (BBa_I15008 + B1006)
 +
 +
L23+L21 = PcyA + terminator (BBa_I15009 + B1006)
 +
 +
|}
 +
Describe your results and mistakes and measure the DNA concentration with the Nanodrop and note the results.
 +
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| L22+L21 a-h=ho1+term
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA conc. ng/µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 260/280
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 260/230
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| L22+L21 a
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 78
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1,8
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1.3
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| L22+L23 d
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 31,7
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2,01
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2,09
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| L22+L23 f
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 30,4
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2,58
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| -11
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| L22+L23 g
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 84,8
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1,98
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2,58
 +
 +
|}
 +
Nothing grew in b,c,e,h
 +
 +
 +
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| L23+L21 a-h=PcyA+term
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA conc. ng/µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 280/230
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 260/230
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| L23+L21 a
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 87,1
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1,95
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2,62
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| L23+L21 b
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 101,1
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1,85
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2,71
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| L23+L21 c
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 31,4
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2,91
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 3,40
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| L23+L21 d
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 184,1
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1,5
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 0.98
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| L23+L21 e
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 98
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1,94
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2,72
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| L23+L21 g
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 49,3
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2,16
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 3,12
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| L23+L21 h
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 122,3
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1,94
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2,77
 +
 +
|}
 +
Nothing grew in f
 +
 +
 +
* Labeled: L22+21 a-h, L23+L21 a-h, strored: pink rack -20 freezer
==<span style="color:orange;">Lysis cassette</span>==
==<span style="color:orange;">Lysis cassette</span>==
Line 40: Line 265:
==<span style="color:grey;">Precipitator</span>==
==<span style="color:grey;">Precipitator</span>==
-
===NAME OF YOUR EXPERIMENT===
+
'''PCR'''
 +
 
 +
 
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name: Ruediger
 +
 
 +
 
 +
 
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 19.07.11
 +
 
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Experiment (Date)
 +
 
 +
PCR 18.07 (Name)
 +
 
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: GFP Pbd
 +
 
 +
|}
 +
PCR-Mixture for one Reaction:
 +
 
 +
For a 50 µl reaction use
 +
 
 +
 
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 32,5µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| H<sub>2</sub>0
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 10µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 5x Phusion Buffer
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| of Primer
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2.5µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Primer fw
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| P18, P19, P20
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2.5µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Primer dw
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| P28
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| dNTPs
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| of Template DNA
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA-Template
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| PCR product of P1,P3,S14 from yesterday
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 0.5 µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Phusion (add in the end)
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 
 +
|}
 +
What program do you use?
 +
 
 +
One set of probes was prepared and then split into 3 tubes each to test them at Annealingtemperatures 44C, 52C and 60C for the first 10 cycles. Then Annealingtemperature 60C for 25 cycles
 +
 
 +
 
 +
To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.
 +
 
 +
 
 +
How did you label the PCR-Product, where is it stored and what do you do next?
 +
 
 +
S14+P18+P28
 +
 
 +
S14+P19+P28
 +
 
 +
S14+P20+P28
 +
 
 +
Stored in PCR product box
 +
 
 +
[[File:Freiburg11_Phusion_ladder.JPG]]
 +
[[File:Freiburg11_7_20_2011_2_48_01_PM.Jpg]]
 +
 
 +
Lane1
 +
Quick Load Marker
 +
Lane2
 +
44C S14+P18+P28
 +
Lane3
 +
44C S14+P19+P28
 +
Lane4
 +
44C S14+P20+P28
 +
Lane5
 +
52C S14+P18+P28
 +
Lane6
 +
52C S14+P19+P28
 +
Lane7
 +
52C S14+P20+P28
 +
Lane8
 +
60 S14+P18+P28
 +
Lane9
 +
60 S14+P19+P28
 +
Lane10
 +
60 S14+P20+P28
 +
 
 +
 
 +
 
 +
 
 +
'''Digestion'''
 +
 
 +
 
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name: Ruediger
 +
 
 +
 
 +
 
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 19.07
 +
 
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Experiment (Date) 19.07 PCR
 +
 
 +
(Name) Ruediger
 +
 
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name:
 +
 
 +
GFP Pbd
 +
 
 +
|}
 +
Procedure
 +
 
 +
 
 +
# add H<sub>2</sub>O (38μl-DNA )
 +
# 5 μl NEB4 buffer  (stored at iGEM’s, -20°C)
 +
# 5 μl 10x BSA  (used 1:10 diluted sample stored at iGEM’s, -20°C)
 +
# DNA (500 ng)
 +
# 1 μl restriction enzymes  (stored at iGEM’s, -20°C)
 +
# heat for 1-2 hours 37°C (6 hours if time)
 +
# heat for 20 minutes 80°C (inactivation of enzymes)
 +
# keep at 4°C if you cannot continue
 +
 
 +
Vector (ratio 1:3 to insert)
 +
 
 +
Inserts (500ng)
 +
 
 +
 
 +
 
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''Components'''
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Vector (μl)
 +
| colspan="3"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Insert1 (μl)
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA (500ng)
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 5,6
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 12,5
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 4,7
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 3,5
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 3,8
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| BSA (100x) (5μl)
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 
 +
|-
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| NEB4 Buffer (5μl)
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 
 +
|-
 +
| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Enzyme 1 (1μl)
 +
| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| SpeI
 +
| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| SpeI
 +
| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| XbaI
 +
| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| XbaI
 +
| style="border-top:none;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| XbaI
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Enzyme 2 (1μl)
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| PstI
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| PstI
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| PstI
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| PstI
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| PstI
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| H<sub>2</sub>O (38 μl- DNA)
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 32,5
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 25,5
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 33,7
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 34,5
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 34,2
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| In total 50 μl
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border-top:0.0069in solid #00000a;border-bottom:0.0069in solid #00000a;border-left:0.0069in solid #00000a;border-right:none;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 
 +
|}
 +
Measured DNA-concentration with Nanodrop to calculate the volume of DNA to do the digestion:
 +
 
 +
 
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Sample
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA concentration (μg/μl)
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| S14+P20+P28 (short:20)
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 132
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| S14+P19+P28 (short 19)
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 145
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| S14+P18+P28 (short 18)
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 107
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| S39
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 90
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| S43
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 40
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 
 +
|}
 +
'''Documentation:'''
 +
 
 +
Why are you doing this experiment? Where are the samples stored? Name of samples, antibiotica resistance, vector used etc.
 +
 
 +
 
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Want to ligate GFPpbd (3 different versions P18/19/20) into PR vectors (one with strong Promotor strong RBS, one with middle Promotor, middle RBS)
 +
 
 +
CM Resistance
 +
 
 +
Mistake: took 100x BSA instead of 10X
-
'''Investigators: NAME'''
+
|}
 +
{{:Team:Freiburg/Templates/footer}}

Latest revision as of 00:58, 22 September 2011


This is the wiki page
of the Freiburger student
team competing for iGEM 2011.
Thank you for your interest!