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Revision as of 12:04, 24 September 2012

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

Component	Volume
Tango buffer 10x	10 µl
SpeI (RE)	5 µl (50 units)
DNA	up to 30 µg
water	ad 150 µl

Incubate for 8 hours at 37°C.
After 3 hours add 2 µl SpeI.
Add 7 µl XbaI and incubate for another 8 hours.

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

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

Adjust to pH 7.0.

Agar-plates

Solve 16g Agar-Agar in 1l LB buffer and boil until solution is clear.
If it is nearly cold pour it into some petri dish.

SOB medium

Component	Volume
Trypton	20,0 g
yeast-extract	5,0 g
NaCl	0,5 g
250mM KCl	10ml
water MiliQ	1l

Solve the components in 1l water.
autoclave
After autoclaving add 5ml MgCl2

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]

@@ Line 100: / Line 100: @@
 | water || ad 150 µl
 |}
-# Incubate for 8 hours at 37°C. After 3 hours add 2 µl SpeI.
+# Incubate for 8 hours at 37°C.
+# After 3 hours add 2 µl SpeI.
 # Add 7 µl XbaI and incubate for another 8 hours.

Team:Tuebingen/NotebookProtocols