Notes

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1. Protein Expression (PYP)

Requirement

        Chemicals:    Bio-Tryptone, Yeas Extract, NaCl, ultrapure Water, 1N NaOH, Agar, Kanamycin, EtOH,         

                            Thermometer, SOC media, Chromophore (4-Hydroxy Cinnamic Acid), IPTG, 

        Others:         Competent Cells BL21*DE3, DNA (Plasmid), 

        Apparatus:    Pasteur Pipette, Measuring Cylinder, 1 Liter Beaker, Magnetic Stirrer, pH meter, Water bath, 

                            Petridish, eppendorf, Pipette (25ml, 1 ml, 10 ml), Parafilm, Aluminium foil, Inoculating loop, Spatula, 

                            Butter paper, 

        Instruments:  Fumehood, Autoclave, Incubator, shaker, Oven, UV, Centrifuge, 

        Buffer:           1 x TAE buffer (pH = 7.5), 

A. Preparation of LB Media

        Lysogeny Broth (LB), a nutritionally rich medium, is primarily used for growth of bacteria. 

B. Protein Purification (PYP)

Requirement

        Chemicals:    Bio-Tryptone, Yeas Extract, NaCl, ultrapure Water, 1N NaOH, Agar, Kanamycin, EtOH,         

                            Therm

References:                   Molecular Cloning, a Laboratory Manual E. F. Fritsch, & J. Sambrook T. Maniatis (Authors)

2. Determination of DNA concentration by Spectrophotometric Estimation

    Experiments where we use DNA, it is often necessary to know its concentration (Units of mass per volume). Most popular method of determining DNA concentration is through spectrophotometric analysis. Since the nitrogenous bases of DNA absorb UV light at 260 nm. 

    Rule of thumb is the concentration of pure double stranded DNA with an absorbance at 260 nm of 1.0 is 50 micro gram per ml. 

    Thus, one can use the formula given below to determine the DNA concentration of a solution.

    Unknown Sample (micro gram per ml)/ Measured Absorbance at 260 nm = 50 (micro gram per ml) x 1.0 (A260)

Reformulate the above formula since there is a linear relation between absorbance and DNA concentration

    Unknown Sample (micro gram per ml) = 50 (micro gram per ml) x  Measured Absorbance at 260 nm x dilution factor

OR

Suppose A260 1.0 is for 50 (micro gram) for 1 ml solution

            so, 1.0 -------- 50 (micro gram) ----------- 1000 (micro liter) solution

            so, 1.0 -------- 50 x 10-3 (micro gram) ----------- 1 (micro liter)

            so, 1.0 x Y (A260) -------- Y x 50 x 10-3 (micro gram) ----------- 1 (micro liter)

            so, 1.0 x Y (A260) -------- Y x 50 (nano gram) ----------- 1 (micro liter)

Now, Find the concentration of Stock solution from which solution was made and Y was measured!

            Suppose 5 micro liter was diluted to 150 micro liter then calculation should be 

            Stock Soln. x 5 (micro liter) = Y x 50 (nano gram) x 150 (micro liter)

Therefore, 

            Stock Soln. = Y x 50 (nano gram) x 150 (micro liter)/  5 (micro liter)

                              = nano gram/micro liter (UNIT)

Unknown Sample (micro gram per ml) = 50 (micro gram per ml) x  Measured Absorbance at 260 nm x dilution factor

                                                       = micro gram/milli liter (UNIT)

Measured Absorbance at 260 nm = Y (A260)

Dilution Factor = 150/5 = 30

NOTE: Careful about the units of calculations

        Online DNA Concentration Calculator 

3. NMR and processing of NMR Data!

    A. Running gNfhsqc on Varian 500 MHz at 20 oC (PYP)

Varian 500 MHz NMR magnet: The Varian 500 MHz NMR system consists of a highly homogeneous superconducting magnet (500 MHz, 1H, 11.7 Tesla), housed within a ultra low-loss helium cryostat with a nominal room-temperature bore of 54 mm.

Temperature Setup:

Important: Make sure that one don't go above the boiling point or below the freezing point of solvent used in sample preparation. 

    1. The temperature that is displayed on the instrument console and on the Vnmr screen display is not necessarily the actual temperature. 

    2. 

    Make sure that NMRpipe is installed on your PC and you have access to this program. First make sure that you have all the files on the right path. 

1. Use the command "varian" or "bruker" to read the *.fid file. eg. Processing of HSQC (1H, 15N) using varian command (Please see the screenshot below).

Here in my case (present study) the varian command reads Acquisition Mode wrongly in y-axis which need to be corrected as States in place of Rance-Kay.

        Primer Design can be done either DNA-based or Protein-based. For my purpose I usually do Protein-based. In this case one has to enter a mutation within the protein sequence encoded by your template DNA, in which case PrimerX generates mutagenic primers based on all possible DNA sequences that can encode the desired mutation, taking into account codon degeneracy. This is recommended for changing a specific amino acid residue into another. 

5. Universal Buffer

    1. (Combination of CAPS, CHES, Bicine, HEPES, PIPES, MES, and Sodium Citrate)

    2. (Combination of Potassium Phosphate, sodium acetate, BIS-TRIS, HEPES, TAPS, CHES, and CAPS)

CAPS    N-cyclohexyl-3-aminopropanesulfonic acid    pKa = 10.4

CHES    N-Cyclohexyl-2-aminoethanesulfonic acid    pKa = 9.3

Bicine    2-(Bis(2-hydroxyethyl)amino)acetic acid    pKa = 8.35

MES    2-(N-morpholino)ethanesulfonic acid        pKa = 6.15 

MOPS    3-(N-morpholino)propanesulfonic acid    pKa = 7.2

CAPSO

HEPES    4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid    pKa = 7.5

PIPES    piperazine-N,N′-bis(2-ethanesulfonic acid    pKa = 6.76

Tris    2-Amino-2-hydroxymethyl-propane-1,3-diol    pKa = 8.07

TAPS    3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonic acid    pKa = 8.44

Sodium Acetate    pKa = 4.76

6. Tris Acetate Buffer (TAE Buffer, pH = 7.5)

Strength and other contents of buffer:

    0.04 M Tris-acetate

    0.001 M EDTA

    100 mM NaCl

So, To make 2L solution of 25xTAE Buffer

Information:

        For 1M Tris-acetate = 242 gm is required

        For 25 mM EDTA = 18.612 gm is required

        For 2.5 M NaCl = 192.2 gm is required

Mix all above three in less than 2L ultrapure water and adjust the pH = 7.5 then make up the final volume to 2L.

M.W (Tris-Acetate) = 121.4 gm

M.W (EDTA) = 372.24 gm

M.W (NaCl) = 58.44 gm

For 1x TAE Buffer of 1L volume,

        Take 40 ml of 25xTAE solution and add 960 ml of ultrapure water = 1L solution of 1xTAE buffer (pH = 7.5)

7. LYSIS Buffer (pH - 8.00

Strength and other contents of buffer:

Authors does not claim any responsibility of results related to above mentioned method. It is the sole responsibility of user whether he/she wants to use the above mentioned information for his/her experiment. Everyone is free to use the information mentioned above with due credit. It is an attempt to share the experiences which I gains everyday!

"Education is a progressive discovery of our ignorance"

- Will Durant

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