Team:Tuebingen/NotebookProtocols

From 2012.igem.org

(Difference between revisions)
(Protocols)
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Mix all reagents and incubate at 22°C for 1h.
Mix all reagents and incubate at 22°C for 1h.
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=== Transformation ===
+
=== Chemotransformation ===
 +
{| class="wikitable"
 +
|-
 +
! Component !! Volume
 +
|-
 +
| chemo-competent ''E. coli'' || 100µl
 +
|-
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| plasmid DNA || up to 10µl (max. 1/10 of volume)
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|}
 +
# 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 at least for 1 hour.
=== Restriction digest ===
=== Restriction digest ===
=== PCR ===
=== PCR ===

Revision as of 14:40, 20 September 2012



Contents

Protocols

Chemo-competent cells

pGEM Ligation

Ligation for TA-cloning of PCR products

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

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

Ligation

Ligation for digested parts and vectors.

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

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

Chemotransformation

Component Volume
chemo-competent E. coli 100µl
plasmid DNA up to 10µl (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 at least for 1 hour.

Restriction digest

PCR