Difference between revisions of "Team:Aix-Marseille/Protocols"

(→‎Monarch PCR and DNA cleanup Kit)
(→‎Monarch PCR and DNA cleanup Kit)
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All centrifugation steps ~13 rpm in a typical microcentrifuge. This ensures all traces of buffer are eluted at each step.  
 
All centrifugation steps ~13 rpm in a typical microcentrifuge. This ensures all traces of buffer are eluted at each step.  
−
#A starting sample volume of 50 μl is recommended. For smaller samples, nuclease-free water can be used to adjust the volume. Add 100 μl DNA Cleanup Binding Buffer to the 50 μl sample.  
+
#A starting sample volume of 50 μL is recommended. For smaller samples, nuclease-free water can be used to adjust the volume. Add 100 μL DNA Cleanup Binding Buffer to the 50 μL sample.  
−
#Insert column into collection tube, load sample onto column and close the cap. Spin for 1 minute, then discard flow-through.
+
#Insert a column into a collection tube, load sample onto the column and close the cap. Spin for 1 minute, then discard flow-through.
−
#Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discard flow-through.
+
#Re-insert column into the collection tube. Add 200 μL DNA Wash Buffer and spin for 1 minute. Discard flow-through.
−
#Repeat Step 5. This step is recommended for removal of enzymes that may interfere with downstream applications (e.g., Proteinase K).  
+
#Repeat Step 5. This step is recommended for removal of enzymes that may interfere with downstream applications.  
 
#Spin for 1 minute.  
 
#Spin for 1 minute.  
 
#Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to the next step.
 
#Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to the next step.
−
#Add 15μl of hot water (70°C) to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute the DNA.
+
#Add 15 μL of hot water (70°C) to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute the DNA.
 
#Repeat step 6.
 
#Repeat step 6.

Revision as of 20:16, 13 August 2018

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Protocols

DH5α competent cells preparation

  1. The day before the preparation, make overnight starters with 100 µL of bacterial cells diluted in 5 mL of LB in a 50 mL Erlenmeyer.
  2. Prepare buffers 1 and 2 and put them on ice. Put Eppendorf tubes and 50 mL Falcon tubes, and p1000 tips at -80°C for future use.
  3. The next day, recover your starters and seed 1/60 bacterial cells in 200 mL of LB, then incubate at 37°C and 180 rpm.
  4. Keep measuring the OD600. When it's between 0,4 and 0,6, recover your cells an pool them in cold 50 mL Falcon tubes.
  5. Centrifuge tubes at 4000 rpm and 4°C for 10 minutes.
  6. Discard supernatant and resuspend each Falcon tube in 20 mL of buffer 1. Afterward, centrifuge at 3500 rpm and 4°C for 5 minutes.
  7. Discard supernatant and resuspend each Falcon tube in 8 mL of buffer 2.
  8. Take 100 µL aliquots in Eppendorf tubes and stock them at -80°C.
Buffer 1 preparation
Product For 40 mL
KAc (1M) 1.2 mL
MnCl2 (0.5M) 4 mL
KCl (1M) 4 mL
CaCl2 (0.1M) 4 mL
Glycerol (80%) 7.5 mL
H2O 19.3 mL
Buffer 2 preparation
Product For 8 mL
KCl (1M) 800 µL
MOPS (1M) 400 µL
CaCl2 (0.1M) 6 mL
Glycerol (80%) 1.5 mL
H2O 500 µL

Transformation

  1. Recover the needed number of competent cells tubes, adding an additional negative control tube.
  2. Add DNA (1µL) into the competent cells tubes.
  3. Incubate tubes on ice for an hour.
  4. Incubate tubes at 42°C for 2 minutes for thermal choc.
  5. Leave tubes on ice for 5 minutes.
  6. Leave tubes at room temperature for 5 minutes.
  7. Add 1 mL of LB per tube, then incubate for 1 hour at 37°C and 180 rpm.
  8. Spread cells on Petri dishes and incubate overnight at 37°C.


Monarch PCR and DNA cleanup Kit

All centrifugation steps ~13 rpm in a typical microcentrifuge. This ensures all traces of buffer are eluted at each step.

  1. A starting sample volume of 50 μL is recommended. For smaller samples, nuclease-free water can be used to adjust the volume. Add 100 μL DNA Cleanup Binding Buffer to the 50 μL sample.
  2. Insert a column into a collection tube, load sample onto the column and close the cap. Spin for 1 minute, then discard flow-through.
  3. Re-insert column into the collection tube. Add 200 μL DNA Wash Buffer and spin for 1 minute. Discard flow-through.
  4. Repeat Step 5. This step is recommended for removal of enzymes that may interfere with downstream applications.
  5. Spin for 1 minute.
  6. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to the next step.
  7. Add 15 μL of hot water (70°C) to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute the DNA.
  8. Repeat step 6.