Team:Tuebingen/NotebookProtocols
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Protocols
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) |
- Add plasmid DNA to cell culture.
- Incubate for 30 min on ice.
- Heat shock for 90 sec at 42°C.
- Add 900 µl LB.
- 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 |
- 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
SOB medium
Genaxxon Plasmid DNA Purification Mini Prep Kit
Genaxxon Gel Extraction Mini Prep Kit
Genaxxon PCR DNA Purification Mini Prep Kit