Monthly Archives: December 2017

Ribonuclease A (RNase A) from bovine pancreas

Overview

  • CAS Number : 9001-99-4
  • EC Number : 232-646-6
  • Enzyme Commission (EC) Number : 3.1.27.5
  • Synonyms: Pancreatic Ribonuclease, RNase A, Ribonucleate 3′-pyrimidinooligonucleotidohydrolase, Ribonuclease A from bovine pancreas Type I-A
  • Molecular weight: 13.7 kDa
  • Optimal temperature : 60°C (activity range of 15–70°C)

Description

  • Ribonuclease A (RNase A) belongs to an endoribonuclease class of ribonucleases. In contrast to exoribonucleases which cleave/degrade RNA in 3’-5’ direction, endoribonucleases degrade RNA endoribonucleolytically in 5’-3’ direction.
  • RNase A is a digestive enzyme which is secreted by the pancreas to digest RNA. It is abundantly present in the pancreas, therefore, the pancreas is a valuable source for RNase A.
  • Mature bovine pancreatic RNase A only has 124 amino acids with molecular weight 13.7 kDa. It lacks tryptophan amino acid.
  • In contrast to others known members of endoribonuclease, RNase A is not a glycoprotein.
  • RNase A is active under a wide range of reaction conditions (temperature range 15 – 70°C; pH range 6–10). The optimal temperature for its activity is 60°C and optimal pH is 7.6.
  • RNase A is quite stable to both heat and detergents.
  • It cleaves both single-stranded and double-stranded RNA as well the RNA strand in RNA-DNA hybrids at a low salt concentration (0 to 100 mM NaCl). However, it specifically cleaves single-stranded RNA at higher salt concentration (0.3M NaCl or higher)

Preparation of RNase A solution:

  • Generally, a 10 mg/ml RNase A solution is prepared in 10 mM Tris.Cl (pH 7.5) or TE [(10 mM Tris.Cl (pH 7.6), 1 mM EDTA]. The stock is stable for at least 1 year at -20°C.
  • Most suppliers provide molecular biology grade RNase A powder which is free from any DNase activity. Sometimes, a low level of DNase activity in RNase stock solution can be detected which can be easily eliminated by incubating the stock solution in boiling water bath for 5 to 10 min (see protocol). Boiling RNase A solution for a short time does not inactivate RNase A, but is sufficient to inactivate DNase activity.
  • If the RNase A stock is suspected to have high DNase activity, it is recommended to prepare 10 mg/ml stock solution in sodium acetate (pH 5.2), incubate the solution in boiling water bath for 20 – 30 min, then adjust the pH with 1M Tris.Cl (pH 7.5). RNase A is comparatively very stable at low pH (between pH 2.0 – 4.5).

References:

Preparation of resuspension buffer for the isolation of plasmid by alkaline lysis method

Overview:

  • Resuspension buffer is used to resuspend bacterial cells during plasmid isolation by alkaline lysis method. It provides an optimal starting pH (pH 8.0) and an ideal condition for subsequent lysis.
  • Resuspension buffer containing Tris and EDTA is very common.
  • Tris.Cl acts as a buffering agent and maintains the pH of the resuspension buffer 8.0.
  • EDTA  chelates the divalent cations which are released upon bacterial lysis. Divalent cations are required for many enzymatic reactions. EDTA action results in inactivation of many enzymes which may harm plasmid DNA.
  • Resuspension buffer can be supplemented with RNase A which helps to get rid of RNA contamination from the plasmid preparation.

To know more, please read the article: Resuspension buffer (solution I) for isolation of plasmid by alkaline lysis method.

Requirements

  • Reagents
    • 1M Tris.Cl (pH 8.0) solution, autoclaved
    • 0.5 EDTA (pH 8.0) solution, autoclaved
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer (optional)

Composition

  • 25 mM Tris.Cl (pH 8.0)
  • 10 mM EDTA (pH 8.0)

Objective

Preparation of 100 ml of resuspension buffer (solution I)

Preparation

Step 1: To prepare 100 ml of resuspension buffer, take 95.5 ml of deionized / Milli-Q water in a 100 ml measuring cylinder/beaker.
Precaution:
  • Do not mix concentrated stock solutions together. This can cause precipitation.
Step 2: Add 2.5 ml of Tris.Cl (pH 8.0) and 2.0 ml of EDTA (pH 8.0). Mix and transfer to a transparent bottle.
Tip:
  • A transparent bottle can easily be examined for any microbial growth in resuspension buffer.

Storage

The solution can be stored at 4°C for 6 months.

Precaution:
  • Frequently check the presence of any microbial growth in resuspension buffer. Discard if you detect any microbial growth.

Application

Preparation of plasmid DNA by alkaline lysis method

Follow the table to prepare resuspension buffer of various volume.
Reagents / Volume 10 ml 25 ml 50 ml 100 ml
1M Tris.Cl (pH 8.0) 0.25 ml 0.625 ml 1.25 ml 2.5 ml
0.5 M EDTA (pH 8.0) 0.2 ml 0.5 ml 1.0 ml 2.0 ml
Water 9.45 ml 23.625 ml 47.25 ml 95.5 ml

Preparation of RNase A containing resuspension buffer for the isolation of plasmid by alkaline lysis method

Overview:

  • Resuspension buffer is used to resuspend bacterial cells during plasmid isolation by alkaline lysis method. It provides an optimal starting pH (pH 8.0) and an ideal condition for subsequent lysis.
  • Resuspension buffer can be supplemented with RNase A. RNase A is a very stable enzyme and is active under the very stringent condition including high alkaline condition, the presence of detergents and chelating agents (EDTA, CDTA).
  • RNase A digest RNAs which are released from bacteria during lysis step, thus allow plasmid preparation free from RNA contamination. However, such plasmid preparation cannot be used for in-vitro transcription due to contamination of RNases.

To know more, please read the article: Resuspension buffer (solution I) for isolation of plasmid by alkaline lysis method.

Requirements

  • Reagents
    • 1M Tris.Cl (pH 8.0) solution, autoclaved
    • 0.5 EDTA (pH 8.0) solution, autoclaved
    • 10 mg/ml RNase A
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer (optional)

Composition

  • 25 mM Tris.Cl (pH 8.0)
  • 10 mM EDTA (pH 8.0)
  • 100 μg/ml RNase A

Objective

Preparation of 100 ml of resuspension buffer (solution I)

Preparation

Step 1: To prepare 100 ml of resuspension buffer, take 94.5 ml of deionized / Milli-Q water in a 100 ml measuring cylinder/beaker.
Precaution:
  • Do not mix concentrated stock solutions together. This can cause precipitation.
Step 2: Add 2.5 ml of Tris.Cl (pH 8.0), 2.0 ml of EDTA (pH 8.0), and 1 ml RNase A. Mix and transfer to a transparent bottle.
Tip:
  • A transparent bottle can easily be examined for any microbial growth in resuspension buffer.

Storage

The solution can be stored at 4°C for 3 – 6 months.

Precaution:
  • Frequently check the presence of any microbial growth in resuspension buffer. Discard if you detect any microbial growth.

Application

Preparation of plasmid DNA by alkaline lysis method

Follow the table to prepare resuspension buffer of various volume.
Reagents / Volume 10 ml 25 ml 50 ml 100 ml
1M Tris.Cl (pH 8.0) 0.25 ml 0.625 ml 1.25 ml 2.5 ml
0.5 M EDTA (pH 8.0) 0.2 ml 0.5 ml 1.0 ml 2.0 ml
10 mg/ml RNase A 0.1 ml 0.25 ml 0.5 ml 1.0 ml
Water 9.45 ml 23.625 ml 47.25 ml 94.5 ml

Preparation of glucose and RNase A containing resuspension buffer for the isolation of plasmid by alkaline lysis method

Overview:

  • Glucose-containing resuspension buffer is used to resuspend bacterial cells during plasmid isolation by alkaline lysis method.
  • Glucose is added to make the resuspension buffer isotonic. However, isotonicity is not required for cell wall containing bacteria including E. coli DH5α. Cell wall containing bacteria can withstand a wide range of solution concentration.
  • Glucose-containing resuspension buffers are prone to microbial growth, therefore cannot be stored for a long time, and need to be kept at 4°C.
  • Resuspension buffer can be supplemented with RNase A. RNase A is a very stable enzyme and is active under the very stringent condition including high alkaline condition, the presence of detergents and chelating agents (EDTA, CDTA).
  • RNase A digest RNAs which are released from bacteria during lysis step, thus allow plasmid preparation free from RNA contamination. However, such plasmid preparation cannot be used for in-vitro transcription due to contamination of RNases.

To know more, please the read article: Preparation of resuspension buffer (solution I) for isolation of plasmid by alkaline lysis method.

Requirements

  • Reagents
    • 1M Glucose solution, filter sterilized
    • 1M Tris.Cl (pH 8.0) solution, autoclaved
    • 0.5 EDTA (pH 8.0) solution, autoclaved
    • 10 mg/ml RNase A
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer (optional)

Composition

  • 50 mM Glucose
  • 25 mM Tris.Cl (pH 8.0)
  • 10 mM EDTA (pH 8.0)
  • 100 μg/ml RNase A

Objective

Preparation of 100 ml of resuspension buffer (solution I)

Preparation

Step 1: To prepare 100 ml of resuspension buffer, take 89.5 ml of deionized / Milli-Q water in a 100 ml measuring cylinder/beaker.
Precaution:
  • Do not mix concentrated stock solutions together. This can cause precipitation.
Step 2: Add 5 ml of 1 M glucose solution, 2.5 ml of Tris.Cl (pH 8.0), 2.0 ml of EDTA (pH 8.0), and 1 ml RNase A. Mix and transfer to a transparent bottle.
Tip:
  • A transparent bottle can easily be examined for any microbial growth in resuspension buffer.

Storage

The solution can be stored at 4°C for 3 – 6 months.

Precaution:
  • Frequently check the presence of any microbial growth in resuspension buffer. Discard if you detect any microbial growth.

Application

Preparation of plasmid DNA by alkaline lysis method

Follow the table to prepare resuspension buffer of various volume.
Reagents / Volume 10 ml 25 ml 50 ml 100 ml
1M Glucose solution 0.5 ml 1.25 ml 2.5 ml 5 ml
1M Tris.Cl (pH 8.0) 0.25 ml 0.625 ml 1.25 ml 2.5 ml
0.5 M EDTA (pH 8.0) 0.2 ml 0.5 ml 1.0 ml 2.0 ml
10 mg/ml RNase A 0.1 ml 0.25 ml 0.5 ml 1.0 ml
Water 8.95 ml 22.375 ml 44.75 ml 89.5 ml

Preparation of glucose containing resuspension buffer for the isolation of plasmid by alkaline lysis method

Overview:

  • Glucose-containing resuspension buffer is used to resuspend bacterial cells during plasmid isolation by alkaline lysis method.
  • Glucose is added to make the resuspension buffer isotonic. However, isotonicity is not required for cell wall containing bacteria including E. coli DH5α. Cell wall containing bacteria can withstand a wide range of solution concentration.
  • Glucose-containing resuspension buffers are prone to microbial growth, therefore cannot be stored for a long time, and need to be kept at 4°C.

To know more, please read the article: Resuspension buffer (solution I) for isolation of plasmid by alkaline lysis method.

Requirements

  • Reagents
    • 1M Glucose solution, filter sterilized
    • 1M Tris.Cl (pH 8.0) solution, autoclaved
    • 0.5 EDTA (pH 8.0) solution, autoclaved
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer (optional)

Composition

  • 50 mM Glucose
  • 25 mM Tris.Cl (pH 8.0)
  • 10 mM EDTA (pH 8.0)

Objective:

Preparation of 100 ml of resuspension buffer (solution I) containing glucose

Preparation:

Step 1: To prepare 100 ml of resuspension buffer, take 90.5 ml of deionized / Milli-Q water in a 100 ml measuring cylinder/beaker.
Precaution:
  • Do not mix concentrated stock solutions together. This can cause precipitation.
Step 2: Add 5 ml of 1 M glucose solution, 2.5 ml of Tris.Cl (pH 8.0) and 2.0 ml of EDTA (pH 8.0). Mix and transfer to a transparent bottle.
Tip:
  • A transparent bottle can easily be examined for any microbial growth in resuspension buffer.

Storage

The solution can be stored at 4°C for 6 months.

Precaution:
  • Frequently check the presence of any microbial growth in resuspension buffer. Discard if you detect any microbial growth.

Application:

Plasmid isolation from E.coli by alkaline lysis method

Follow the table to prepare resuspension buffer of various volume.
Reagents / Volume 10 ml 25 ml 50 ml 100 ml
1M Glucose solution 0.5 ml 1.25 ml 2.5 ml 5 ml
1M Tris.Cl (pH 8.0) 0.25 ml 0.625 ml 1.25 ml 2.5 ml
0.5 M EDTA (pH 8.0) 0.2 ml 0.5 ml 1.0 ml 2.0 ml
Water 9.05 ml 22.625 ml 45.25 ml 90.5 ml

Resuspension buffer (solution I) for isolation of plasmid by alkaline lysis method

  • Resuspension buffer (solution I) is used for the isolation of plasmid DNA by alkaline lysis method. Bacterial cells, obtained from the culture (liquid culture or colonies, grown on a agar plate), is resuspended in this buffer. The purpose of resuspension buffer is to provide an optimal starting pH (pH 8.0) and an ideal condition for subsequent lysis.
  • The classical composition of resuspension buffer (designed by Birnboim and Doly) contained Lysozyme, Glucose, Tris.Cl, and CDTA (or EDTA). Most of the recent formulations do not contain lysozyme and glucose.
  • Lysozymes are glycoside hydrolases that destroy bacterial cell walls by catalyzing the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan. However, for most bacteria including E. coli DH5α, lysis solution was found to induce complete lysis, thus eliminated the use of lysozymes.
  • Glucose is added to make the solution isotonic. However, isotonicity is not required for cell wall containing bacteria including E. coli DH5α. Cell wall containing bacteria can withstand a wide range of solution concentration. Therefore, Glucose is not included in the recent recipes of resuspension buffer. Furthermore, glucose-containing resuspension buffer cannot be stored for a long time, and need to be kept at 4°C.
  • EDTA (or CDTA) chelates the divalent cations which are released upon bacterial lysis. Divalent cations are required for many enzymatic reactions. EDTA action results in inactivation of many enzymes which may harm plasmid DNA.
  • Tris.Cl acts as a buffering agent and maintains the pH of the resuspension buffer 8.0.
  • Now researchers prefer to supplement resuspension buffer with RNase A. RNase A is a very stable enzyme and is active under the very stringent condition including high alkaline condition, the presence of detergent and chelating agent (EDTA). Addition of RNase A in resuspension buffer helps to remove RNA from the plasmid preparation. In the subsequent lysis step, RNase A digests the RNA of the bacteria. However, such plasmid preparation cannot be used for in-vitro transcription due to contamination of RNases. In addition, RNase A containing resuspension buffer should be stored at 4°C and has a limited life (3 – 6 months).
  • pH indicator, LyseBlue from Qiagen, can also be added to the resuspension buffer. LyseBlue ensures the complete lysis and subsequently neutralization step. Both steps are very important to get high quality of plasmid DNA.
  • Resuspension buffer is not included in the protocol of plasmid isolation using plasmid isolation kit provided by some manufacturers (see Zyppy Plasmid Miniprep Kit). In those procedures, highly concentrated lysis buffer is added directly to the overnight grown liquid culture of bacterial cells.
  • The following types of resuspension buffer can be used for plasmid isolation

Preparation of freezing medium containing DMSO and FBS

Overview:

  • Freezing medium can be used to preserve cell lines for long term at ultra-low temperature. This method of preserving cell line is called cryopreservation.
  • An ideal freezing medium must allow 100% recovery without causing any change in cell characteristics after revival.
  • Freezing medium is nothing but a complete medium supplemented with high concentration of serum and a cryoprotective agent such as DMSO or glycerol.
  • As the name suggest, cryoprotective agents protect cells from lysis due to ice crystal formation at temperature below freezing point.
  • Serum concentration as high as 90% can be used in freezing medium. High serum concentration improves cell viability and recovery after thawing.
  • Serum-free chemically-defined freezing media are also available. They are very useful to preserve those cell lines which are maintained in serum-free chemically defined medium.
  • Both serum-containing and serum-free freezing medium are available commercially.
  • Serum-containing medium is used for cell lines which are maintained in serum-supplemented growth medium. We recommend to check cell line manual for optimal freezing medium.

Requirements

  • Reagents
    • Fetal bovine serum (FBS) or Fetal calf serum (FCS)
    • DMSO (sterile)
    • Complete growth medium (optional)
  • Equipment and disposables
    • 50 ml sterile polypropylene tubes
    • Pipetboy
    • Pipets
    • Laminar flow hood
Note:
  • Use the same growth medium which is used to maintain cell line (e.g., use DMEM, if the cell line is maintained in DMEM medium).

Composition:

  • 50% FBS
  • 40% complete growth medium
  • 10% DMSO

Objective:

Preparation of 50 ml serum-containing freezing medium

Note: All operations must be done under sterile condition. Wipe carefully surface of reagent containing bottles (e.g., DMSO bottle, FBS bottle) with 70% ethanol before placing them inside flow hood.

Preparation:

Step 1: To prepare 50 ml freezing medium, transfer 25 ml FBS and 20 ml growth medium to a 50-ml sterile polypropylene tube. Tighten the cap of the tube and mix by gentle inversion. Store it on ice.

Tips: You can also use sterile containers like beaker. We recommend you to use 50 ml sterile polypropylene tube.

Step 2: Step 2: Add 5 ml DMSO slowly while shaking the tube. Tighten the cap of the tube and mix by inverting the tube many times (5 – 6 times).
Note: Mixing of DMSO to serum is an exothermic reaction which can cause denaturation of serum proteins, resulting in precipitation. Therefore, it is recommended to use chilled FBS.

Precautions: Do not store DMSO on ice. It will solidify on ice.

Step 3 (optional): Check the sterility of freezing by keeping a small aliquot in a petri dish in the CO2 incubator.
Tips: We recommend you to check sterility of freezing medium.

Storage:

Store at 4°C for few days. Freezing medium is stable for at least 6 months at -20°C.

Tips: Freezing and thawing multiple times can cause proteins denaturation and precipitation.

Applications:

  • Serum-containing freezing medium is used for cryopreservation of cell lines.

Follow the table to prepare freezing medium of different volume.

Reagent/volume 10 ml 50 ml 100 ml 250 ml
FBS 5 25 50 125
DMSO 1 5 10 25
Growth medium 4 20 40 100

Cryopreservation of cell culture

  • Cell culture is not static. Cells in culture acquire changes which can be either genetically programmed (e.g., senescence in primary culture) or due to accumulation of genetic abnormalities (mutations, gain or loss of whole chromosomes or part of chromosomes). In addition to this, changes in gene expression pattern and epigenetic modifications due to several reasons including fluctuations in culture condition, contamination, mishandling and stressful condition to culture, can also lead to permanent changes in cell behavior (e.g., stem cell culture can differentiate, or lose its ability to differentiate). Therefore, we need a method to preserve cell culture which stop or slow down these processes.
  • Cryopreservation is an efficient way to preserve cells at ultra-low temperature (below -135°C) which stop all physiological processes and biological aging. It is a routinely used technique in all cell culture laboratories.
  • During preservation at ultra-low temperature, cells die due to many reason including lysis due to ice crystal formation, pH change, dehydration, and alterations in the concentration of electrolytes. Four distinct phases of cell preservation and revival process can cause to damage to cells…………
    • when temperature reduced to above freezing point (hypothermia)
    • when temperature reduced to below freezing point
    • during frozen state
    • during revival
  • Cryopreservation methods ensure that cells are alive at ultra-low temperature and maintain their features when revived after long term frozen state.
  • Most cryopreservation methods rely on
    • cryoprotectants
    • slow cooling
    • rapid revival
  • To cryopreserve cells, cells are suspended in freezing medium, followed by slow cooling and subsequently storage in liquid nitrogen.
  • Freezing medium is nothing but growth medium supplemented with cryoprotectant. Serum containing growth medium contains high amount of serum (upto 90%).
  • Cryoprotectants, the most important component of freezing medium, function by preventing the formation of ice crystals, thus protect cells from lysis.
  • Polyalcohols (e.g., glycerol, ethylene glycol, 2,3 butanediol) and DMSO can be used as cryoprotectants, often a concentration varies from 5 – 20%. Most cryoprotectants have ability to penetrate the cell membrane and function by replacing part of the water in the cell.
  • DMSO is most frequently used cryoprotectant. However, some cells lines are sensitive to DMSO. In such situation, glycerol can be  used. Glycerol is less toxic than DMSO, however, osmotic problem associated with glycerol at the time of thawing restrict its uses.
  • High concentration of serum can also be added in freezing medium. High serum concentration correlate with better survival upon thawing.
  • Serum-free chemically defined freezing medium are also available which are prepared by adding cryoprotectant to serum-free chemically defined medium growth.
  • Serum-containing freezing mediums are used for cell lines growing in serum-supplemented growth medium whereas serum-free freezing medium is used for those cell lines which are maintained in serum-free chemically defined medium.