Laboratory 1:

  • Basic ethics in laboratory work

 

    • Important values in scientific experiment: Art

                        Accuracy, Reproducibility, Traceability

 

    • Labeling and Identification:

Label should have sufficient information and to concise
for both samples and reagents
(ex; ID, concentration, Date, etc)

 

    • Record and Result:

Write own protocol (do not “copy and paste” from original protocol)
Write down any calculation made
Leave record of any changes during experiment
Keep all related data
Make a conclusion at the end of experiment

 

  • Basic Calculations in molecular biology

 

    • Concentration: indicate the amount of sample (solute) in given amount of solvent

w/w, w/v, v/v

      • Simple concentration: absolute amount of solute in given volume of solvent

gram/liter (g/L),  milligram/milliliter (mg/ml)
need to specify the units of both solute and solvent
                                                Ex) 100mg/ml Ampicillin , 100mg of ampicillin in 1ml of water

           

      • Percentage (%):        
  • mass/volume percent (% (w/v)); 

amount of solute in gram in final volume100ml of solution.
Ex) 10% SDS (w/v) : 10g of SDS dissolved in 100ml of water

  • volume percent (% (v/v)): a common expression of a solution's concentration. It is the volume fraction expressed with a denominator of 100, and thus is defined as:

{\textrm {volume\ percent}}={\frac {\textrm {volume\ of\ solute}}{\textrm {volume\ of\ solution}}}\times 100


Volume percent is usually used when the solution is made by mixing two fluids, such as liquids
            Ex) 70% Ethanol (v/v); 70ml of 100% Ethanol + 30ml of diluent (water) =
100ml of final volume

 

      • Molar concentration (Molarity):

       

       

       

Molar concentration or molarity is most commonly expressed in units of moles of solute per liter of solution

Molarity (M) =            # of moles solute          =   moles  = mol/L
                                                                      

                        Volume of solution in liter        V
 
                       moles  =  mass of solute (grams)
                                                                                                molar mass  (Mw or Fw)

 

                       M     =    mass of solute (gram)
                                                                              (Molar mass)  (vol of solution in liter)

 

Problem 1: Calculate the molarity of a solution prepared by dissolving 25 g of common salt in 500 ml of water. (Given: Molar mass of NaCl is 58.44g/mol).

Solution: First, we calculate the number of moles of solute present:
Moles of NaCl = Total mass of NaCl/Molar mass = 25/58.44 = 0.43moles.
Volume in liters = 500 ml/1000 ml = 0.5 L.
From the above formula, we have,
Molarity = Number of moles of NaCl / Volume of water in liters.
= 0.43/0.5

Molarity M = 0.86 mol/L

Mass of solute (g) = M x Molar mass (Fw) x vol of solution (L)

Problem 2: Calculate amount of NaCl needed to make 3M NaCl for 1500ml

                        mass of solute = (M)(Molar mass)(vol of solution)
                                       = (3) (58.44)(1.5)
                                       = 262.98 gram

 

      •  Fold concentration and dilution

In biological experiments, many reagents such as reaction buffers and running buffers are prepared in concentrated form for the convenience of use and preparation.

10X reagent: 10 times concentrated that needs to be diluted 10x (fold) to make 1x solution

  • with known volume of final solution: need 1/nX of final volume (final vol/ n time)

 Ex1)   to make 500ml of running buffer with 20X concentrate,
            500ml/20 =25ml and Q.S. to 500ml with diluents)

Ex 2),  Final reaction volume is 20ul, how much volume of 10X reaction buffer?
            20ul/10 =2ul of 10X reaction buffer

  • To dilute fixed volume of concentrate to lower concentration:

           diluting by nX (fold):   1 part concentrate + (n-1) parts diluent
           (Ex,     2-fold dilution,  1 part concentrate +1 (2-1) parts diluent
                    5-fold dilution,  1 part concentrate +4 (5-1) parts diluents

  • To calculate volume of concentrate to fixed volume of diluents (sample):

           Volume of concentrate = Volume of diluents sample/(n-1)

           (Ex,  adding 5X sample buffer to 20ul of sample
                    20ul/ (5-1) =20ul/4 = 5ul of 5X sample buffer)

 


  • Lab 1: Making Culture Media

 

    • Luria Broth (LB medium) for 1 liter
    •   Bacto-tryptone  10g
        Yeast Extract 2g
        NaCl 5g
        NaOH pellet 2 pellet
        ddH2O Q.S (Quantum Sufficit) to 1 liter

 

 

    • LB Agar medium (1.5% Agar in LB) for 1 liter
    •   Bacto-Agar  15g for 1 liter of LB medium

 

 

    • Preparation: (work as 2 people / group)
      • Prepare 1 liter of LB medium

 

    •   Bacto-tryptone      g
        Yeast Extract     g
        NaCl     g
        NaOH pellet     pellet
        ddH2O Q.S (Quantum Sufficit) to      liter

 

 

 

      • Prepare two 300 ml of LB-agar medium

Aliquot   300ml of LB medium into two 500ml flasks
Add   Bacto-agar    __________ g to each flasks

 

      • Aliquot 60ml of LB into two 250ml flasks

            Aliquot the remaining LB medium  (~80ml) in 100ml bottle

                                                              

      •  Autoclave them at 121oC for 20 min in liquid cycle

 
 

      • Prepare Ampicillin Stock solution (100mg/ml)

Measure 100mg of Ampicillin
Dissolve in 1ml of ddH2O
Sterilize by sterile filter (syringe and filter disk)
Store on ice or freezer

 

      • Preparing LB agar plate (each individual)
        • LB-agar medium

After autoclave, cool it down to ~60oC  
(touch with hand and it will feel a little hot)

pour ~25ml of  LB-agar into petri-dish

 

        • LB-agar with Ampicillin

Add Ampicillin stock solution (100mg/ml) by 1000x dilution

add _________ Ampicillin stock solution into cooled 300 ml LB-agar 

pour ~25ml of  LB-agar into petri-dish

 

      • Streaking out from frozen cell stock

Purpose: to select a single colony (which is coming from a single cell)
               By sequentially diluting out the concentrated cells
               Start with ~0.05 X 108  --, Be sure to flame the streaking loop each time
           

 

 

 

 

 


           Streak MC4100 (E.coli, F-DlacU169araD136relArpsL150flbB5301deoC7ptsF25thi)
           on LB-agar plate (2 plates) and LB-agar ampicillin plate
            incubate at 37oC o/n

 

 

 

 

 

 

 

 

 

 

 

 

 


This information is given as a guide to the student attending the Bio4905 laboratory as a means to review some of the information. It is not meant to replace the laboratory. No emphasis as to what will be required of the student is given in this text, indeed information that is given in the these transcripts may make little sense if the student has not first attended the relevant laboratory.

 


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