Cloning

• Plan construction

plasmid according to host organism/selection marker/copy number/etc.
order primers for insert (if needed)
(SnapGene Software is nice)

• Prepare insert (PCR) and clean it up

• Restriction Digest of plasmid and insert
  

1-2ug DNA
1ul of each restriction enzyme
2ul buffer (FastDigest)
ddW up to 20ul

• Blunting (optional)
  

Add 1ul of Klenow(it will fill 5’ and remove 3’) and 1ul of 2mMdNTP to restriction mix and incubate 15 min at 37C

• To prevent plasmid ligation on itself add 1ul of CIAP/other alkaline phosphatase after restriction of plasmid and incubate 1h 37C (optional)
  
• Gel purification of fragments (run agarose gel with fresh TAE buffer, cut fragment with clean knife then use DNA purification kit)
  
• Ligation with Instant Ligase Master Mix (NEB)
  

Combine 20-100ng of plasmid and insert (1:1, 1:3 or 3:1 molar ratios)
Add ddW up to 4ul
Add 4ul of Blunt or Sticky end Ligation Mix and mix by pipeting
Keep on ice for 10min (for blunt ends keep 15min at Troom and then place on ice)

• Bacterial Transformation

Bacterial Transformation
Competent cells preparation (Rubidium Chloride protocol)

•  Incubate colony of bacteria overnight in LB/SOC 37c shaker.
• Dilute 1:100 in YT media (16g bacto tryptone,10g bacto yeast extract, 5g NaCl, pH7.0 (with NaOH5N) – for 1L)

•  Grow to OD 0.4-0.6 3-5h
  
•  Spin cells 4C 5000g 10min
  
• Remove media
  
•  Add 100ml TFB1
  

Rubidium Chloride 100mM
Manganese Chloride 50mM
Potassium Acetate 30mM
Calcium Chloride monohydrate 10mM
Glycerol 15%
pH5.8 with acetic acid
filter sterilize, 4C before use

• resuspend (pipete)
  
•  Incubate on ice 5min
  
• Spin cells 4C 5000g 10min
  
• Add 5-10ml TFB2
  

MOPS 10mM
Rubidium Chloride 10mM
Calcium Chloride 75mM
Glycerol 15%
pH6.5 with KOH
Filter sterilize, 4C before use

• resuspend
  
• Incubate on ice 20-60min
  
• Freeze -80C in 1.5ml tubes (200ul)

Transformation

• Thaw competent cells on ice 15min
  
• Mix DNA (plasmid/ligation mix) with 100ul competent cells
  
• Incubate 30min on ice
  
• Heat shock 42C 45sec-1min
  
• Add 1ml LB
  
• Incubate 1h 37C with shaking
  
• Plate on LB with appropriate antibiotic

DNA precipitation 

·         Reagents:
• 3 M sodium acetate pH 5.2 or 5 M Ammonium or Sodium acetate
• DNA solution
• 100% ethanol (ice cold) or -20 degree Celsius
• 70% ethanol kept on 4 degree Celsius

·         Procedure:
1. Measure the volume of the DNA sample.
2. Add 1/10 volume of sodium or potassium acetate, pH 5.2
 - These amounts assume that the DNA is in TE only; if DNA is in a solution containing salt, adjust salt accordingly to achieve the correct final concentration.
3. Mix well vortex.
4. Add 2 volumes of ice cold 100% ethanol.
5. Mix well by vortex.
6. Place on ice or at -80 degrees C for 30 minutes. Or at -20 overnight.
7. Spin a maximum speed in a microfuge 10-15 min.
Check for DNA pellet little opaque type
8. Carefully decant supernatant.
9. Add 500ul 70% ethanol. Mix gentely. Carefully decant supernatant. Wash step
10. Air dry pellet.
11. Resuspend pellet in the appropriate volume of TE or water once is fully dried.