Team:Tuebingen/NotebookProtocols

From 2012.igem.org

(Difference between revisions)
(→LB medium)
(→Protocols)
Line 21: Line 21:
Mix all reagents in a 0.5 ml tube. Incubate reaction at 4°C over night.
Mix all reagents in a 0.5 ml tube. Incubate reaction at 4°C over night.
 +
 +
Line 40: Line 42:
|}
|}
Mix all reagents and incubate at 22°C for 1 hour.
Mix all reagents and incubate at 22°C for 1 hour.
 +
 +
Line 56: Line 60:
# Add 900 µl LB.
# Add 900 µl LB.
# Let the bacteria grow at 37°C for at least 1 hour.
# Let the bacteria grow at 37°C for at least 1 hour.
 +
 +
== Restriction digest ==
== Restriction digest ==
 +
 +
=== control digest ===
=== control digest ===
Line 76: Line 84:
|}
|}
# Incubate at least for 1 hour at 37°C.
# Incubate at least for 1 hour at 37°C.
 +
 +
=== preparative double digest ===
=== preparative double digest ===
 +
 +
 +
=== plasmid linearization ===
=== plasmid linearization ===
 +
 +
== PCR ==
== PCR ==
Line 100: Line 115:
| water || 36 µl
| water || 36 µl
|}
|}
 +
'''PCR conditions'''
'''PCR conditions'''
Line 120: Line 136:
| 6 || (hold) || 4°C
| 6 || (hold) || 4°C
|}
|}
 +
 +
Line 137: Line 155:
|}
|}
Adjust to pH 8.5.
Adjust to pH 8.5.
 +
'''Gel'''
'''Gel'''
Line 148: Line 167:
|}
|}
Solve agarose in TAE 1x buffer and boil until solution is clear.
Solve agarose in TAE 1x buffer and boil until solution is clear.
 +
'''Well loading'''
'''Well loading'''
Line 159: Line 179:
|}
|}
Can be scaled up linearly.
Can be scaled up linearly.
 +
 +
== LB medium ==
== LB medium ==
Line 176: Line 198:
|}
|}
Adjust to pH 7.0.
Adjust to pH 7.0.
 +
 +
== SOB medium ==
== SOB medium ==
 +
 +
== Genaxxon Plasmid DNA Purification Mini Prep Kit ==
== Genaxxon Plasmid DNA Purification Mini Prep Kit ==
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/Plasmid-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/Plasmid-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]
 +
 +
== Genaxxon Gel Extraction Mini Prep Kit ==
== Genaxxon Gel Extraction Mini Prep Kit ==
[http://www.genaxxon.com/catalogue/DNA-Purification-Kits/PCR-and-Gel-extraction-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]
[http://www.genaxxon.com/catalogue/DNA-Purification-Kits/PCR-and-Gel-extraction-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]
 +
 +
== Genaxxon PCR DNA Purification Mini Prep Kit ==
== Genaxxon PCR DNA Purification Mini Prep Kit ==
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/PCR-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/PCR-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]
 +
 +
== QIAGEN Plasmid Midi Kit ==
== QIAGEN Plasmid Midi Kit ==
[http://www.qiagen.com/products/plasmid/qiagenplasmidpurificationsystem/qiagenplasmidmidikit.aspx#Tabs=t2 Manual provided by QIAGEN]
[http://www.qiagen.com/products/plasmid/qiagenplasmidpurificationsystem/qiagenplasmidmidikit.aspx#Tabs=t2 Manual provided by QIAGEN]

Revision as of 09:58, 24 September 2012



Protocols

Contents

Chemo-competent cells

pGEM Ligation

Ligation for TA-cloning of PCR products

Component Volume
2X Rapid Ligation Buffer 5 µl
pGEM vector 0.5 µl (25ng)
PCR product 3.5 µl
T4 DNA ligase 1 µl (3 Weiss units)

Mix all reagents in a 0.5 ml tube. Incubate reaction at 4°C over night.



Ligation

Ligation for digested parts and vectors

Component Volume
10X T4 DNA Ligase Buffer 1 µl
vector DNA 1 µl (20-100 ng)
insert DNA 5 µl (up to 5:1 molar ratio insert to vector)
T4 DNA ligase 1 µl (1 unit)
water 2.5 µl

Mix all reagents and incubate at 22°C for 1 hour.



Chemotransformation

Component Volume
chemo-competent E. coli 100 µl
plasmid DNA up to 10 µl (max. 1/10 of volume)
  1. Add plasmid DNA to cell culture.
  2. Incubate for 30 min on ice.
  3. Heat shock for 90 sec at 42°C.
  4. Add 900 µl LB.
  5. Let the bacteria grow at 37°C for at least 1 hour.



Restriction digest

control digest

Component Volume
Tango buffer 10x 1 µl
XbaI (RE) 0.5 µl (5 units)
SpeI (RE) 0.5 µl (5 units)
DNA 1 µl (up to 1 µg)
water 7 µl
  1. Incubate at least for 1 hour at 37°C.


preparative double digest

plasmid linearization

PCR

Component Volume
Taq/Pfu buffer 5 µl
Taq/Pfu polymerase 1 µl
primer forward 0.5 µl (100 pmol/µl)
primer reverse 0.5 µl (100 pmol/µl)
dNTPs 2.5 µl (200 µM)
template DNA 1 µl
water 36 µl


PCR conditions

Step Duration Settings
1 2 min 94°C
2 45 sec 94°C
3 30 sec gradient or annealing temperature
4 90 sec 72°C
steps 2-4: 30 cycles
5 7 min 72°C
6 (hold) 4°C



Gel electrophoresis

TAE buffer 50x

Component Volume
0.05 M EDTA 18.61 g
1 M acetic acid 60.05 g
2 M Tris 242.28 g
water 1 l

Adjust to pH 8.5.


Gel

Component Volume
TAE 1x buffer 120 ml
Agarose 1.2 g

Solve agarose in TAE 1x buffer and boil until solution is clear.


Well loading

Component Volume
PCR product or DNA 5 µl
Loading dye 6x 1 µl

Can be scaled up linearly.


LB medium

incl. Agarplatten

Component Volume
Trypton 10,0 g
yeast-extract 5,0 g
NaCL 5,0 g
water 1,0 l

Adjust to pH 7.0.


SOB medium

Genaxxon Plasmid DNA Purification Mini Prep Kit

[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/Plasmid-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]



Genaxxon Gel Extraction Mini Prep Kit

[http://www.genaxxon.com/catalogue/DNA-Purification-Kits/PCR-and-Gel-extraction-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]



Genaxxon PCR DNA Purification Mini Prep Kit

[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/PCR-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]



QIAGEN Plasmid Midi Kit

[http://www.qiagen.com/products/plasmid/qiagenplasmidpurificationsystem/qiagenplasmidmidikit.aspx#Tabs=t2 Manual provided by QIAGEN]

Retrieved from "http://2012.igem.org/Team:Tuebingen/NotebookProtocols"