Team:IIT Madras/Protocols

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Protocols

PCR Protocol

Composition of PCR reaction:

• PHusion Phusion® High-Fidelity DNA Polymerase(from Finnzymes) - 0.2 mul
• 5x Phusion HF Buffer (from Finnzymes) - 4 mul
• dNTP – 1 mul
• DNA Template – 1 mul
• Primers(from Bioserve) – 1 mul each.
• Water – 12.8 mul

Total volume for the reaction is 20 mul. Program used:

1. 98 C for 2min.
2. 98 C for 30sec.
3. (Tm+3)C for 30sec.
4. 72C for x sec(15sec per kb).
5. GOTO 2 30 times.
6. 72C for 30 min.
7. 4C for storage.

Digestion Protocol

Composition of the digestion mixture:

• DNA - 4 mul
• Enzyme 1 - 1.2 mul
• Enzyme 2 - 1.2 mul
• Buffer - 2 mul
• Water - 11.6 mul

Total volume for the reaction is 20 mul.

Steps:

1. For EcorRI, PstI – The reaction mixture is kept at 37 degrees for 1.5 hours. For other Enzymes – The reaction mixture is kept at 37 degrees for 5 hours.
2. Inactivate enzyme by heating at 80 degrees for 20 mins.

T4 Ligation Protocol

Composition of Ligation mixture:

• T4 Ligase Buffer - 1mul
• 6:1 molar insert:vector (vector~10ng)
• miliQ water - (8.5 - DNA volume) mul
• T4 ligase – 0.5 mul

Steps:

1. Leave reaction at 22.5degC for 30min
2. Denaturate ligase at 65degC for 10min
3. Store at -20degC

Ultracompetent Cell Preperation Protocol

1. Materials/Buffers * SOB SOLUTION FOR COMPETENT CELL PREPARATION
   1. 0.5% yeast Extract
   2. 2% Tryptone
   3. 10mM NaCl
   4. 2.5mM KCl
   5. 10mM MgCl2
   6. 10mM MgSO4.
   7. Dissolve all in nanopure water and autoclave

            * TRANSFORMATION BUFFER FOR COMPETENT CELL PREPARATION
   

   1. 10mM PIPES
   2. 15mM CaCl2
   3. 250mM KCl
   4. Dissolve in nanopure water and adjust pH to 6.7 with KOH or HCl. Then add MnCl2 to 55mM and adjust final volume. Sterilize by filtration with 0.45 µm filter. Store at 4C
2. Cells were cultured on LB agar plate overnight at 37C.
3. 10-12 colonies were cultured in 250ml SOB medium.
4. It was incubated at 37C for 1hour. Then the flasks were transferred to 19C. It was incubated till the OD600 reached 0.5
5. Flask was placed in ice for 10min.
6. The cells were pelleted by spinning at 4000rpm for 10min at 4C.
7. Cells were resuspended in 80ml ice cold TB(Transformation Buffer) and stored on ice for 10min.
8. It was centrifuged again at 4000rpm for 10min at 4C.
9. Pellet was resuspended in 20ml of TB with 1.5ml DMSO.
10. Final volume was aliquoted into microcentrifuge tubes (100-500µl) and stored at -80C

CAUTION!

   * Caution: The whole procedure after the cells are pelleted out needs to be carried out in ice.
   * Caution: TB buffer is heat sensitive, never take it out of ice.

Transformation Protocol

1. 100µl competent cells were thawed on ice
2. 2 µl Plasmid DNA added to the tube and shaken gently.
3. Mixture left on ice for 30 min.
4. Heat shock given at 42C for 2min.
5. Incubated on ice for 3-5 min.
6. 800 µl of LB broth added.
7. Flasks were shaken at 37C for 1hr.
8. They were centrifuged at 3000rpm for 5min and the pellet was resuspended into 100ul of the supernatant.
9. The 100 µl of the transformation mix was plated on LB agar plates.
10. Plates were incubated at 37C overnight.

Miniprep Protocol

1. Overnight cultures were harvested (2-3ml broth cultures).
2. They were centrifuged at 13000rpm for 1min.
3. The pellet was resuspended in 250 µl of HP1 solution.
4. The cells were lysed by adding 250 µl of lysis solution i.e. HP2. Tubes were inverted 5-6 times.
5. 350 µl of neutralization solution i.e. HN3 was added. Tbes were inverted 5-6 times to mix the solutions.
6. They were centrifuged at 13000rpm for 10 mins to get a white pellet.
7. The supernatant was carefully transferred to a HiElute Miniprep spin column.
8. It was centrifuged at 13000rpm for 1 min. Flow through was discarded.
9. 500 µl of wash solution i.e. HPB was added to the column.
10. It was centrifuged at 13000rpm for 1 min. Flow through was discarded.
11. 700 µl of wash solution i.e. HPE was added to the column.
12. It was centrifuged at 13000rpm for 1 min. Flow through was discarded.
13. It was centrifuged at 13000rpm for 1 min.
14. The column was transferred to a fresh tube.
15. 50 µl of elution buffer was added carefully to the center of the column.
16. Incubate for 1 min
17. It was centrifuged at 13000rpm for 1 min by placing it in a fresh tube.

PCR Protocol with Taq Polymerase

1. Mix contains:
   1. 0.4 µl Taq polymerase.
   2. 2 µl Bffer (10X).
   3. 0.8 µl dNTPs.
   4. 0.4 µl forward Primer.
   5. 0.4 µl backward Primer.
   6. 0.8 µl Template.
   7. 15.2 µl MilliQ water.
2. Program used:
   1. 96C for 2 min.
   2. 96C for 30sec.
   3. (Tm-5)C for 30sec.
   4. 72C for ‘x’min (1min per kb).
   5. GOTO 2 30 times.
   6. 72C for 30min.
   7. 4C for storage.

   Purification by Phenol/CHCl3 Method Kept for restrction digestion at 37 degC for 16hrs and purify by phenol/CHCl3 method
   

   1]50uL mixture -  make the volume to 100uL
   2]add 100uL of phenol/CHCl3 – conc?
   3]centrifuge 20,000 for 5 min
   4]aqueous layer (approx. 100uL)
   5]add 1/10 volume of 3M sodium Acetate (10uL)
                      +
     2.5 volumes of 95% ethanol (250uL)
   6]incubate at -20/-40degC for 2 hrs
   7]centrifuge 20,000xg for 20 min at 4 degC
   8]pellet washed with 70% ethanol
   9]air dry the pellet
   10]dissolve pellet in 11uL MQ
                  \/
          \/              \/
          10uL            1uL load on gel and estimate its concentration
   
   
   restriction digestion of vector 
   as conc. Increases ....
   gel purification.
   100uL [chk] kept for restiction
   
   extract gel using gel extraction kit
   
   quantify RE vector by running agarose gel
   1:1      3:1    5:1

   
   

   Vector  
   Insert  
   T4 DNA ligase   
   Buffer  
   MQ      
   Total            10uL
   Length of insert [in bases] X ng of vector X (ratio)
   lenght of vector [bases]
                          = ng of insert needed for [[ratio] Molar basis]

   
   

   
   
   after 16 + 1 hr
   

   Purification Of Ligation Mixture
   [using 2M potassium acetate pH=8]
   
   1] Both rxn mixture (1:3 & 1:5) was pooled (20uL)
      make up the ligation mixture volume to 50uL
   2] add 6.25uL of 2M K-acetate (pH 8)
   3] add 2.5 volume of 95% ethanol (125uL) – mix well
   4] allow the ppt to form (-20degC, 2 hrs)
   5] centrifuge at 14000 rpm for 10min in eppendorf
   6] wash the pellet twice with 70% ethanol (125uL)
      10 min , 14000 rpm (twice)
   7] air dry to remove alcohol completely
   8] resuspend the pellet in 5uL of deionozed water (sterile)
   9] transfer the whole mixture for electroporation
   10] transform the (40uL) cells with 5uL of ligation mix. (run a control also)
   11]immediately add 1mL of media without antibiotic
   12] incubate for 1.5 hr
   13] centrifuge 5000rpm for 1 min
   14] remove 800uL of supernatant
   15] remaining 200uL, resuspend cells
   16] plate in selection plate
   

   Electroporation
   

   
   

   1]   add 40uL cells in electroporation cuvette 
                      +
        1uL DNA (or 5uL ligation mixture)
   2]   keep the cuvette on ice
        use biorad gene pulser 2000V, 25uF, 200ohms
        pulse approx. 4.5 to 5msec
   3]   add 1mL G-M17B +20mM MgCl2 + 2mM CaCl2 medium
   4]   keep the cuvette for 5min on ice
   5]   incubate 1 to 1.5 hr @ 30degC
   6]   centrifuge 1 min,   10,000rpm
   7]   remove 800 uL of media
   8]   resuspend in 200uL of media
   9]   spread plate on G-M17B + 1.5% agar + anti-biotic (10ug/ml)
      containing glucose 5g/l, 20 mM MgCl2 & 2mM CaCl2

   
   
   
   
   
   Preparation of electro competent cells of L.lactis NZ9000
   

   DAY 1:
   incubate 10mL culture in 10ml G-SGM17B media with -80degC stock grow
   at 30 degC
   
   DAY 2:
   add 0.5ml culture in 10 ml  G-SGM17B
   
   grow at 30 degC
   
   DAY 3:
   dilute the 10ml culture in 90ml of  G-SGM17B media
   
   grow till OD600 (0.2 to 0.3)  approx. 3Hr[it should not go above 0.3]
   
   centrifuge 10min at 5,000 rpm(4degC)
   
   Wash cells with 40-45mL sucrose / 10% glycerol (4deg C)
   
   Centrifuge (20-30mins, 5k rpm)
   keep cells on ice for 15 mins in 50ml of 0.5M sucrose
   10% glycerol / 0.05M EDTA (4degC)
   
   centrifuge 20-30 min , 5,000 rpm
   
   wash cells with 25ml 0.5M sucrose /10% glycerol(4degC)
   
   centrifuge 20min,  5,000rpm
   resuspend the cells in 0.8-1ml 0.5 sucrose /10% glycerol(4degC)
   
   use 40uL per electroporation [keep on ice]
   
   keep cells in aliquots -80degC
   let them defreeze on ice before each use
   

   
   
   
   
   
   Reagents for electro competent cells prep
   [1] G-SGM17 (100mL) (M17 + 0.5M sucrose + 2.5% Glycine + 0.5% glucose)
   

   2 X M17= 50ml
   sucrose  = 17.115g
   glycine = 2.5g
   20% glucose = 2.5 ml                    {volume made to 100ml}

   Note:add sucrose and glycine to M17 and sterilize by autoclaving
   

      add sterile glucose when cooled
   
   

   
   [2] glucose stock = 20% - autoclave
   
   [3] 1M sucrose
   

     sucrose=34.2g<br>
          deionized water= make volume to 100ml<br>

   
   [4] Glycerol 50%
   

      glycerol=50ml<br>
          deionized water= 50ml<br>

   
   [5] 0.5M sucrose - 10% Glycerol soln.
   

      1M sucrose =100ml<br>
          50% glycerol= 40ml<br>
          deionozed water= 60ml<br>
                                  ==200ml<br><br>

   [6] 0.5M sucrose - 10% Glycerol soln. -0.05M EDTA
   

      1M sucrose =50ml
          50% glycerol= 20ml
          disodium EDTA = 1.86g
                  \/
          heat to dissolve
                  \/
          deionized water= make volume 100ml

   
   [7] 0.5M CaCl2 (50ml)
   

      CaCl2.2H2O= 3.676g
          water = make vol 50ml

   
   [8] 1M MgCl2 (50ml)
   

      MgCl2.6H2O = 10.165g
          deionized water = make up the vol to 50ml

   
   [9] revival medium - GM17CaMg
   

      2 X M17= 5ml
          0.5M CaCl2 = 0.04ml
          1M MgCl2 = .2ml
          20% Glucose = 0.25ml
          deionized water = 4.5ml
                          ==10ml

   
   [10] plate for selection
   

      2 X M17 agar medium = 200ml
          20% Glucose = 10ml
          0.5M CaCl2 = 1.6ml
          1M MgCl2 = 8ml
          water = 180ml
          chloramphenicol = 400ul
                          == 400ml

   [11] G5M17 -Cml plate
   
   [12] G5M17 -agar plate
   
   [13] G5M17 medium (5X 10ml)
   
   also to be autoclaved MQ water, tubes, measuring cylinder.
   

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