Team:KU Leuven/Protocols

From 2013.igem.org

(Difference between revisions)
Line 59: Line 59:
#Inoue transformation buffer
#Inoue transformation buffer
{| class="wikitable"
{| class="wikitable"
-
| Reagent || Final concentration (mM)  || Amount per liter  || align="right" |
+
| Reagent || Final concentration (mM)  || Amount per liter   
|-
|-
-
| MnCl2  || 55  || 10.88g (from MnCl2*4H2O)    || align="right" |
+
| MnCl2  || 55  || 10.88g (from MnCl2*4H2O)     
|-
|-
-
| CaCl2 || 15  ||  2.20g (from CaCl2*2H2O)    || align="right" |
+
| CaCl2 || 15  ||  2.20g (from CaCl2*2H2O)     
|-
|-
-
| KCl || 250  ||  18.65g (from KCl)    || align="right" |
+
| KCl || 250  ||  18.65g (from KCl)     
|-
|-
-
| PIPES || 10  ||  20ml (from 0.5M stock solution)     || align="right" |
+
| PIPES || 10  ||  20ml (from 0.5M stock solution)    
|-
|-
-
| H2O || to 1 liter  ||      || align="right" |
+
| H2O || to 1 liter  ||       
|}
|}

Revision as of 10:06, 12 July 2013

Secret garden

Congratulations! You've found our secret garden! Follow the instructions below and win a great prize at the World jamboree!


  • A video shows that two of our team members are having great fun at our favourite company. Do you know the name of the second member that appears in the video?
  • For one of our models we had to do very extensive computations. To prevent our own computers from overheating and to keep the temperature in our iGEM room at a normal level, we used a supercomputer. Which centre maintains this supercomputer? (Dutch abbreviation)
  • We organised a symposium with a debate, some seminars and 2 iGEM project presentations. An iGEM team came all the way from the Netherlands to present their project. What is the name of their city?

Now put all of these in this URL:https://2013.igem.org/Team:KU_Leuven/(firstname)(abbreviation)(city), (loose the brackets and put everything in lowercase) and follow the very last instruction to get your special jamboree prize!

tree ladybugcartoon


Contents

Chemically competent E.coli cells

Method 1: Short CaCl2 method (source: Veerle)

Procedure

Perform every action on ice – also resuspending your cells !! Do not shock freeze (liqN2) – just transfer from ice to -80°C !!! Work sterile

  • Inoculate 3ml growth medium with your cells of choice (DH5alpha or TOP10 for plasmid maintenance & cloning)
  • Grow overnight at 37°C with sufficient aeration
  • Inoculate 100 ml LB with 1 ml of overnight culture
  • Grow at 37°C to an Optical Density600nm of approx 0.5 to 0.8 (usually 2-3 hrs)
  • Centrifuge cells (3700-4000 rpm 4°C 12min – sterile 50ml tube)
  • Re-suspend pellet on ice with FSB to 15 ml (cold) for each 100 ml pellet
  • Incubate cells 10min on ice
  • Centrifuge cells (3700 – 4000 rpm 4°C 10min)
  • Re-suspend pellet on ice in 4-8 ml FSB (cold) for each 100 ml pellet
  • Aliquot cells appropriately (200-400 µl aliquots) and freeze aliquots at -80°C

Buffers and solutions

  1. Growth medium
    • LB 25g/l
  1. FSB
    • 10 mM Potassium Acetate
    • 10% glycerol
    • 10 mMKCl
    • 50 mM CaCl2
    • Check pH – must be around 6.2 – if need be adjust with AcAc (HCl) or KOH
    • Buffer should be filter-sterilized (0.45 micrometer filter) but has been autoclaved also

Inoue method (source: Sambrook, Maniatis)

Procedure

Perform every action on ice – also resuspending your cells !! Work sterile

  • Pick a single colony from a freshly transformed plate (after overnight growth @ 37 degrees)
  • Transfer the colony to 25 ml growth medium in a 250 ml erlenmeyer (sterile !)
  • Incubate the culture @ 37°C for 6 – 8 hrs under vigourous shaking (250 – 300 rpm)
  • Prepare 3 1L flasks with 250 ml growth medium in each
  • Inoculate the flasks with 10, 4 or 2 ml of the dayculture -> you create 3 different starting optical densities.
  • Incubate the cultures @ 18-22°C overnight under moderate shaking (180 – 220 rpm)
  • Monitor the OD600nm until it reaches 0.55
  • Place cells in an ice-water bath to cool them down quickly (-> swirl occasionally, keep them in for approx 10min)
  • Harvest cells @4°C for 10min at 2500g
  • Pour off supernatant (to biological waste) – make sure all remaining droplets are removed
  • Resuspend gently (swirl !) in 80 ml icecoldinoue transformation buffer
  • Harvest cells @4°C for 10min at 2500g
  • Pour off supernatant (to biological waste) – make sure all remaining droplets are removed
  • Resuspend gently (swirl !) in 20 ml icecoldinoue transformation buffer
  • Add 1.5 ml 100% DMSO – mix by swirling
  • Store whole on ice for approx 10 minutes
  • Aliquot as quickly as possible 100 – 200 microliter aliquots into 1.5 ml tubes (precooled on ice) and snapfreeze them into a liqN2 bath

Buffers and solutions

  1. Growth medium
  2. Inoue transformation buffer
Reagent Final concentration (mM) Amount per liter
MnCl2 55 10.88g (from MnCl2*4H2O)
CaCl2 15 2.20g (from CaCl2*2H2O)
KCl 250 18.65g (from KCl)
PIPES 10 20ml (from 0.5M stock solution)
H2O to 1 liter
Retrieved from "http://2013.igem.org/Team:KU_Leuven/Protocols"