Team:Peshawar/Experiments

iGEM Peshawar

Experiments

Our Lab Protocols

Things required:

  1. 1X TAE Buffer
  2. Agarose
  3. EtBr

Protocol:

Calculate the amount of agarose required for 50 ml TAE Buffer based on percentage of gel required.

2 % Agarose Gel:

  1. 50 ml 1X TAE Bufeer
  2. 1.0 g Agarose
  3. 0.5 ul EtBr

Mix thoroughly and then pour in gel caster and attach comb. Let it solidify for 30 – 40 minutes. 

Things required:

  1. Nuclease Free Water
  2. Yeast
  3. Tryptone
  4. NaCl
  5. Agar

100ml Broth Preparation:

  1. NF Water                                100ml
  2. Yeast                                       0.5 g
  3. Tryptone                                  1.0 g
  4. NaCl                                        1.0 g

Mix all these in a beaker and autoclave for 20 minutes at 121OC and 15 psi.

100ml Agar Preparation:

  1. NF Water                                100 ml
  2. Yeast                                       0.5 g
  3. Tryptone                                  1.0 g
  4. NaCl                                        1.0 g
  5. Agar                                        1.0 g

Mix all these in a beaker and autoclave for 20 minutes at 121OC and 15 psi. 

ANTIBIOTIC

STOCK SOLUTION

WORKING SOLUTION

SOLUTION MAKING

amp

100 mg/ml

100 ug/ml

ddH2O

chl

25 mg/ml

25 ug/ml

EtOH

ken

50 mg/ml

50 ug/ml

dH2O/ddH2O

tet *light sensitive*

10 mg/ml

10 ug/ml

dH2O/ddH2O

Protocol:

  1. Resuspend E. coli / Plasmid cells by shaking
  2. Label 2 1.5 ml Eppendorf tubes.
  3. Transfer 1000ul of E. coli / Plasmid (overnight suspension)
  4. Close caps – then spin for 1 minute to pellet the cells.
  5. Discard supernatant – drain off completely.
  6. Add 100ul of ice-cold GTE solution to each tube – resuspend by pipetting up and down till it is homogeneous.
  7. Add 200 ul of SDS/NaOH solution to each tube – close caps – mix rapidly inverting tubes 5 times.
  8. Stand tubes on ice for 5 minutes.
  9. Add 150 ul of ice-cold KOA solution – close caps – mix rapidly inverting 5 times. (precipitate will be made)
  10. Stand tubes on ice for 5 minutes.
  11. Spin for 5 minutes to pellet the precipitate.
  12. Transfer 400 ul of supernatant from each tube into clean 1.5 ml Eppendorf tubes.
  13. Add 400 ul of isopropanol to each tube of supernatant – close cap – invert 5 times.
  14. Stand at room temperature for only 2 minutes.
  15. Spin for 5 minutes.
  16. Discard supernatant.
  17. Add 200 ul of 100% ethanol. 

Things required:

  1. Sample culture
  2. Resuspension buffer (B1)
  3. Lysis buffer (B2)
  4. Neutralization buffer (B3)
  5. Wash buffer 1
  6. Wash buffer 2
  7. Elution buffer

Protocol:

  1. Take 5 ml of culture in an Eppendorf tube.
    1. Centrifuge at 13000 rpm for 30 seconds.
    2. Take pellet.
    3. Repeat numerous times for desired amount of culture.
    4. Mix 200 ul Resuspension buffer (B1) using vortex or pipette.
    5. Add 200 ul of Lysis buffer (B2) and gently invert 5 – 6 times, then incubate at room temperature for 1 minute. Color should change to purplish pink. DO NOT VORTEX.
    6. Add 400 ul of Neutralization buffer (B3) and gently invert till the color changes to yellow. Precipitate will be formed. Incubate at room temperature for 2 minutes.
    7. Centrifuge at 13000 rpm for 5 minutes.
    8. Collect supernatant in the column provided with the kit.
    9. Centrifuge for 1 minute, and discard the flow through.
    10. Add 200 ul Wash buffer 1 and then centrifuge for 1 minute. Discard the flow through.
    11. Add 400 ul Wash buffer 2 and then centrifuge for 1 minute. Discard the flow through.
    12. Transfer the contents of the flow through to a clean 1.5 ml Eppendorf tube.
    13. Add 30 ul Elution buffer.
      1. Incubate for 1 minute at room temperature.
      2. Centrifuge for 1 minute.
      3. Discard the column.
      4. Mark the Eppendorf tube and place the plasmid DNA at -20OC for further use. 

Reaction volume: 25 ul

Three main steps:

  1. Initial Denaturation:                            95OC for 5 minutes
    1. Denaturation:                          95OC for 30 seconds
    2. Annealing:                              65OC for 15 seconds
    3. Extension:                               68OC for 30 seconds – 90 seconds (depends on size)
    4. Final Extension:                                  68OC for 5 minutes

 

DNA Polymerase Types:

  1. Taq                                                      1 x
  2. Pyrococcus furiosus                            6 x  
  3. Phusian DNA Polymerase                  50 x
  4. Q5

 

Protocol:

  1. Standard Rxn Buffer                          2.5 ul
  2. dNTPs                                                 0.5 ul
  3. Forward Primer                                   0.5 ul
  4. Reverse Primer                                    0.5 ul
  5. Template DNA                                   1-2 ul
  6. Polymerase Enzyme                           0.125 ul
  7. Nuclease Free Water                           upto 25 ul 

For colony PCR, take a colony of choice from a plate and mix in 10 ul of NF water. Then take    1 - 2 ul of sample from that mixture

 

Reaction volume: 25 ul

Three main steps:

  1. Initial Denaturation:                            95OC for 5 minutes
    1. Denaturation:                          95OC for 30 seconds
    2. Annealing:                              65OC for 15 seconds
    3. Extension:                               68OC for 30 seconds – 90 seconds (depends on size)
    4. Final Extension:                                  68OC for 5 minutes

 

DNA Polymerase Types:

  1. Taq                                                      1 x
  2. Pyrococcus furiosus                            6 x  
  3. Phusian DNA Polymerase                  50 x
  4. Q5

 

Protocol:

  1. Standard Rxn Buffer                          2.5 ul
  2. dNTPs                                                 0.5 ul
  3. Forward Primer                                   0.5 ul
  4. Reverse Primer                                    0.5 ul
  5. Template DNA                                   1-2 ul
  6. Polymerase Enzyme                           0.125 ul
  7. Nuclease Free Water                           upto 25 ul 

Reaction volume: 50 ul

 

  1. SINGLE DIGESTION
    1. Buffer (Cut Smart or 3.1)                               5 ul
    2. Restriction Enzyme                                         1 ul
    3. DNA Sample                                                   1 ul
    4. Nuclease Free Water                                       upto 50 ul  

 

  1. DOUBLE DIGESTION
    1. Buffer (Cut Smart or 3.1)                               6 ul
    2. Restriction Enzyme                                         1+1 ul
    3. DNA Sample                                                   2 ul
    4. Nuclease Free Water                                       upto 50 ul

 

PROCEDURE:

  1. Mix all components in 1.5 ml Eppendorf tube
  2. Place the tube at 37OC for 60 minutes
  3. Then place the tube at 80OC for 20 minutes. This step is called as heat-kill step.
  4. Store the tube at -20OC for further use. 

Reaction volume: 10 ul

 

  1. Single Digestion
    1. Buffer                                                             2.5 ul
    2. Restriction Enzyme                                         1 ul
    3. DNA Sample                                                   3-6 ul (depends on conc. of sample)
    4. Nuclease Free Water                                       upto 10 ul
    5. BSA (if using buffer 2)                                   0.5 ul  

 

  1. Double Digestion
    1. Buffer                                                             4 ul
    2. Restriction Enzyme                                         1+1 ul
    3. DNA Sample                                                   4-6 ul (depends on conc. of sample)
    4. Nuclease Free Water                                       upto 10 ul
    5. BSA (if using buffer 2)                                   0.5 ul

 

Procedure:

  1. Mix all components in 1.5 ml Eppendorf tube
  2. Place the tube at 37OC for 60 minutes
  3. Then place the tube at 80OC for 20 minutes. This step is called as heat-kill step.
  4. Store the tube at -20OC for further use. 

Things Required:

  1. Buffer (NEB)
  2. Enzyme
  3. Insert (Part A, Part B, or both)
  4. Plasmid Backbone/ Vector
  5. Nuclease Free Water

 

Protocol:

  1. Take 4 ul Buffer in a small tube.
  2. Add Insert and Vector in 3:1 molar concentration values.
  3. Add NF water upto 19 ul.
  4. Add 1 ul Ligase enzyme.

Place the tube in Thermal Cycler at 16OC for 60 minutes.

Place it at 80OC for 20 minutes for heat kill.

Mark the tube and store at -20OC for further use. 

Things Required:

  1. Buffer (Invitrogen)
  2. Enzyme
  3. Insert (Part A, Part B, or both)
  4. Plasmid Backbone/ Vector
  5. Nuclease Free Water

 

Protocol:

  1. Take 4 ul Buffer in a small tube.
  2. Add Insert and Vector in 3:1 molar concentration values.
  3. Add NF water upto 19 ul.
  4. Add 1 ul Ligase enzyme.

Place the tube in Thermal Cycler at 25OC for 60 minutes.

Place it at 65OC for 10 minutes for heat kill.

Mark the tube and store at -20OC for further use. 

Protocol:

  1. Inoculate 0.2 ml (200 ul) of overnight culture in 10 ml LB broth.
  2. Put in shaker incubator at 37OC for 2 hours.
  3. Check O.D 600. It should be 0.2 – 0.3
  4. Take a centrifuge tube and place it on ice to cool.
  5. Place the culture on ice for 5 minutes
  6. Transfer it to pre – cool centrifuge tube
  7. Centrifuge at 6000 rpm for 5 minutes at 4OC.
    1. Discard supernatant
    2. Gently resuspend the cells with ice – cold 50 Mm CaCl2
    3. Leave on ice for 40 minutes.
    4. Centrifuge at 6000 rpm for 5 minutes at 4OC.
      1. Discard supernatant.
      2. Gently resuspend with 2 ml CaCl2
      3. Store on ice until needed.
      4. Make aliquots of 100 ul and place at -80OC. 

Things Required:

  1. Competent cells (DH5a)         100 ul
  2. Plasmid DNA                         1 ul
  3. SOC Media (NEB)                 400 ul

 

Protocol:

  1. Thaw the competent cells on ice (-80OC to 4OC)
  2. Add 100 ul of C.C in 1.5 ml Eppendorf tube.
  3. Mix 1 ul of DNA in100 ul of C.C
  4. Incubate on ice for 40 minutes at 4OC.
  5. Give the cells a heat shock for 60 seconds (4OC to 42OC). This is a very critical step.
  6. Incubate on ice for 5 minutes.
  7. Add 400 ul of SOC media in LFH.
  8. Incubate the mixture in incu-shaker at 37OC for two (2) hours.
  9. Plate the cells.
  10. Wrap with parafilm and incubate in incubator at 37OC overnight.