Team:Freiburg/Notebook/14 July

From 2011.igem.org

(Difference between revisions)

Revision as of 10:47, 20 September 2011

Contact

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

red light receptor

3A-assembly: Digest

Investigators: Jakob

3A-assembly of S22 (ho1)+S21a (terminator) and S23 (pcyA)+S21a (terminator)

Digestion

Name:Jakob	Date: 14.07.2011
Project Name: 3A-assembly of pcyA+ terminator, ho1+terminator

Procedure

1. add H2O (16μl-DNA )

2. 2μl NEB4 buffer

3. 2 μl 10x BSA (used 1:10 diluted )

4. DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)

5. 1 μl restriction enzymes

6. heat for 1-2 hours 37°C (6 hours if time)

7. heat for 20 minutes 80°C (inactivation of enzymes)

8. keep at 4°C if you cannot continue

Measured DNA-concentration with Nanodrop to calculate the volume of DNA to do the digestion:

Sample Name	DNA concentration (μg/μl)
Vector pSB1T3	20
terminator	38.8
ho1	176
pcyA	173

Restriction enzymes you need to cut the vector, insert1 and insert 2:

Components	Vector (μl)	Insert1 and 2 (μl)
DNA (μl)	2	Ho1 2.5 pcyA 2.5	Terminator 5.5 terminator 5.5
BSA (10x) (2μl)
NEB4 Buffer (2μl)
Enzyme 1 (1μl)	EcoRI	EcoRI	XbaI
Enzyme 2 (1μl)	PstI	SpeI	PstI
H2O (14 μl- DNA)
In total 20 μl

3A-assembly: Ligation and transformation

Investigators: Julia

Ligation of S22 (ho1)+S21a (terminator) and S23 (pcyA)+S21a (terminator) in vector pSB1T3.

L23: Ligation of S23+S21a in pSB1T3 L22: Ligation of S22+S21a in pSB1T3

Plasmids were transformed in cells and plated out.

PCR

Investigators: Julia

PCR of the red light receptor, cph8.

Primer:

P 10
P 11

DNA template:

BBa_I150010

Lysis cassette

Quickchange PCR

Name: Theo

Date: 14.07.2011

Continue from Experiment Lysis Cassette

Project Name: Correct number of nucleotides between RBS and ATG of temp. sensitive repressor from

Lysis Device

PCR-Mixture for one Reaction:

For a 50 µl reaction use

32,5µl	H₂0	Name
10µl	6x HF Phusion Buffer
2.5µl	Primer fw	P29 (1:10)
2.5µl	Primer dw	P30 (1:10)
1µl	dNTPs
1µl	DNA-Template	M15a (from S15->diluted to 5ng/µl)
0.5 µl	Phusion (add in the end)

What program do you use?

	95°C - 15 sec
95°C - 5 min	55°C - 15 sec	72°C -5 min	16°C - ∞
	72°C - 5’ + 5’’
	20x

Precipitator

Test-digest

Investigators: Rüdiger

Testdigest of the minipreps, Sophie did (june, 13).

Enzyme 1: PstI
Enzyme 2: EcoRI

Image of different samples from miniprep.

@@ Line 10: / Line 10: @@
 A-assembly of S22 (ho1)+S21a (terminator) and S23 (pcyA)+S21a (terminator)
-S21a:
-*Enzyme 1: XbaI
-*Enzyme 2: PstI
-S22, S23
+'''Digestion'''
-*Enzyme 1: EcoRI
-*Enzyme 2: SpeI
-Vector:
-*Enzyme 1: EcoRI
+{| style="border-spacing:0;"
-*Enzyme 2: PstI
+| style="border-top:0.0139in solid #808080;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Name:Jakob
-*Enzyme 3: DpnI
+| style="border:0.0139in solid #808080;padding:0.0194in;"| Date: 14.07.2011
-<br/>
+|-
+| colspan="2"  style="border:0.0139in solid #808080;padding:0.0194in;"| Project Name: 3A-assembly of pcyA+ terminator, ho1+terminator
+|}
+&nbsp;
+Procedure
+&nbsp;
+. add H2O (16μl-DNA )&nbsp;&nbsp;&nbsp;
+. 2μl NEB4 buffer
+. 2 μl 10x BSA &nbsp;&nbsp; &nbsp;(used 1:10 diluted )
+. DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
+. 1 μl restriction enzymes
+. 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
+&nbsp;
+Measured DNA-concentration with Nanodrop to calculate the volume of DNA to do the digestion:
+{| style="border-spacing:0;"
+| style="border-top:0.0139in solid #808080;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Sample Name
+| style="border:0.0139in solid #808080;padding:0.0194in;"| DNA concentration (μg/μl)
+|-
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Vector&nbsp; pSB1T3
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| 20
+|-
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| terminator
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| 38.8
+|-
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| ho1
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| 176
+|-
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| pcyA
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| 173
+|}
+&nbsp;
+Restriction enzymes you need to cut the vector, insert1 and insert 2:
+{| style="border-spacing:0;"
+| style="border-top:0.0139in solid #808080;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| '''Components'''
+| style="border-top:0.0139in solid #808080;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| <center>'''Vector (μl)'''</center>
+| colspan="2"  style="border:0.0139in solid #808080;padding:0.0194in;"| <center>'''Insert1 and 2 (μl)'''</center>
+|-
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| DNA (μl)
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| 2
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Ho1 2.5
+pcyA 2.5
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| Terminator 5.5
+terminator 5.5
+|-
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| BSA (10x) (2μl)
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;
+|-
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| NEB4 Buffer (2μl)
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;
+|-
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Enzyme 1 (1μl)
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;EcoRI
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;EcoRI
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;XbaI
+|-
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Enzyme 2 (1μl)
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;PstI
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;SpeI
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;PstI
+|-
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| H2O (14 μl- DNA)
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;
+|-
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| In total 20 μl
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| &nbsp;
+| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"| &nbsp;
+|}
+&nbsp;
 ===3A-assembly: Ligation and transformation===