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TE Buffer
TE buffer is a commonly used buffer solution in molecular biology, especially in procedures involving DNA or RNA. "TE" is derived from its components: Tris, a common pH buffer, and EDTA, a molecule chelating cations like Mg2+. The purpose
of TE buffer is to protect DNA or RNA from degradation.
Recipe
A typical recipe for making TE buffer is:
* 10 mM Tris, bring to pH 7.5 with HCl
* 1 mM EDTA
Based on nuclease studies from the 1980's, the pH is usually adjusted to 7.5 for RNA and 8.0 for DNA. The respective DNA and RNA nucleases are supposed to be less active at these pH values, but pH 8.0 can safely be used for storage of both DNA and RNA.
EDTA further inactivates nucleases, by binding to metal ions required by these enzymes.
of TE buffer is to protect DNA or RNA from degradation.
Recipe
A typical recipe for making TE buffer is:
* 10 mM Tris, bring to pH 7.5 with HCl
* 1 mM EDTA
Based on nuclease studies from the 1980's, the pH is usually adjusted to 7.5 for RNA and 8.0 for DNA. The respective DNA and RNA nucleases are supposed to be less active at these pH values, but pH 8.0 can safely be used for storage of both DNA and RNA.
EDTA further inactivates nucleases, by binding to metal ions required by these enzymes.
TAE Buffer
50x TAE Buffer (Tris-Acetate-EDTA) for DNA Electrophoresis
242 gm Tris base
57.1 ml Acetic acid
100ml 0.5M EDTA [EDTA is not soluble at acidic pH - adjust pH to 8.0 with 10 M NaOH (and acetic acid if necessary)]
Add ddH2O to 1 liter and adjust pH to 8.5.
Create Blunt-Ended DNA
- Protocol for blunting ends by 3' overhang removal and 3' recessed end fill-in:
DNA should be dissolved in any 1X restriction enzyme NEBuffer or 1X EcoPol Reaction Buffer supplemented with 33μM each dNTP. Add 1 unit Klenow per microgram DNA and incubate 15 minutes at 25°C. Stop reaction by adding EDTA to a final concentration of 10mM and heating at 75°C for 20 minutes. CAUTION: Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times may result in recessed ends due to the 3'→ 5' exonuclease activity of the enzyme. - When DNA Polymerase I, Large (Klenow) Fragment is used to sequence DNA using the dideoxy method of Sanger et al., 1 unit/5 μl reaction volume is recommended.
- Klenow Fragment is also active in any restriction enzyme reaction buffer and T4 DNA Ligase reaction buffer when supplemented with dNTPs.
T4 DNA Ligation Protocol
Description:
Catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids
Application notes:
Room Temperature Ligation:
For convenience, ligations may be done at room temperature (20-25°C).
Alternatively, at 16oC o/n.
For cohesive (sticky) ends, use 1 µl of T4 DNA Ligase in a 20 µl reaction for 10 minutes.
For blunt ends, use 1 µl of T4 DNA Ligase in a 20 µl reaction for 2 hours (or 1 µl high concentration T4 DNA Ligase for 10 minutes. Alternatively, NEB's Quick Ligation Kit (NEB #M2200S, [30 reactions] or NEB #M2200L, [150 reactions]) is uniquely formulated to ligate both blunt and cohesive (sticky) ends in 5 minutes at room temperature).
Q: How much DNA should be used in a ligation using T4 DNA Ligase?
A: The overall concentration of vector + insert should be between 1-10 μg/ml for efficient ligation. Insert:vector molar ratios between 2 and 6 are optimal for single insertions. Ratios below 2:1 result in lower ligation efficiency. Ratios above 6:1 promote multiple inserts. If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios.
Formula for calculating required DNA amounts:
Insert (ng): Insert Size X Vector (ng) * Insert to Vector Molar Ratio / Vector
Size
Catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids
Application notes:
Room Temperature Ligation:
For convenience, ligations may be done at room temperature (20-25°C).
Alternatively, at 16oC o/n.
For cohesive (sticky) ends, use 1 µl of T4 DNA Ligase in a 20 µl reaction for 10 minutes.
For blunt ends, use 1 µl of T4 DNA Ligase in a 20 µl reaction for 2 hours (or 1 µl high concentration T4 DNA Ligase for 10 minutes. Alternatively, NEB's Quick Ligation Kit (NEB #M2200S, [30 reactions] or NEB #M2200L, [150 reactions]) is uniquely formulated to ligate both blunt and cohesive (sticky) ends in 5 minutes at room temperature).
Q: How much DNA should be used in a ligation using T4 DNA Ligase?
A: The overall concentration of vector + insert should be between 1-10 μg/ml for efficient ligation. Insert:vector molar ratios between 2 and 6 are optimal for single insertions. Ratios below 2:1 result in lower ligation efficiency. Ratios above 6:1 promote multiple inserts. If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios.
Formula for calculating required DNA amounts:
Insert (ng): Insert Size X Vector (ng) * Insert to Vector Molar Ratio / Vector
Size
E. Coli Competent Cell Protocol
Overview: There are several ways to prepare competent cells for plasmid DNA transformation. This is the chemical method. Advantages are that it’s simple to complete, requires no special equipment and gives good transformation efficiencies.
Disadvantages are that the efficiency is somewhat lower (vs. electroporation). In general, it is best to use this when the transformation efficiencies is not the problem, otherwise
you might want to use and make the competent cells for electroporation.
Materials:
• Single colony of E. coli cells to be transformed
• LB medium
• 0.1 M CaCl2, ice cold
• LB amp plates
• 42 °C water bath
• 0.1 M CaCl2+15% glycerol, sterile
Procedure:
1. Inoculate one colony from LB plate into 2 ml LB liquid medium. Shake at 37 °C overnight.
2. Inoculate 1-ml overnight cell culture into 100 ml LB medium (in a 500 ml flask).
Shake vigorously at 37 °C to OD600 ~ 0.25-0.3 (usually it takes about 1.5-2 hours).
3. Chill the culture on ice for 15 min. Also make sure the 0.1M CaCl2 solution and 0.1M CaCl2 plus 15% glycerol are on ice.
4. Centrifuge the cells for 10 min at 3300 g (e.g. 4,000 rpm in tabletop centrifuge) at 4 °C.
5. Discard the medium and resuspend the cell pellet in 30-40 ml cold 0.1M CaCl2.
6. Keep the cells on ice for 30 min.
7. Centrifuge the cells as above.
8. Remove the supernatant, and resuspend the cell pellet in 6 ml 0.1 M CaCl2 solution plus 15% glycerol.
9. Pipet 0.4-0.5 ml of the cell suspension into sterile 1.5 ml micro-centrifuge tubes.
Freeze these tubes on dry ice and then transfer them to -70 C freezer.
Notes:
1. The transformation efficiency is about 1-5x106/ul DNA when using the competent cells prepared with this method.
Important: all steps after harvesting the cell should be done on ice (or at 4 °C)
2. The frozen competent cells are stable for 6 months, but once a tube is taken from the freezer and thawed, any unused portion should be discarded.
3. After the competent cells are made, the transformation efficiency should be checked by transformation using plasmid DNA of known concentration.
Reference:
*Current Protocols in Molecular Biology (1.8.2)
Disadvantages are that the efficiency is somewhat lower (vs. electroporation). In general, it is best to use this when the transformation efficiencies is not the problem, otherwise
you might want to use and make the competent cells for electroporation.
Materials:
• Single colony of E. coli cells to be transformed
• LB medium
• 0.1 M CaCl2, ice cold
• LB amp plates
• 42 °C water bath
• 0.1 M CaCl2+15% glycerol, sterile
Procedure:
1. Inoculate one colony from LB plate into 2 ml LB liquid medium. Shake at 37 °C overnight.
2. Inoculate 1-ml overnight cell culture into 100 ml LB medium (in a 500 ml flask).
Shake vigorously at 37 °C to OD600 ~ 0.25-0.3 (usually it takes about 1.5-2 hours).
3. Chill the culture on ice for 15 min. Also make sure the 0.1M CaCl2 solution and 0.1M CaCl2 plus 15% glycerol are on ice.
4. Centrifuge the cells for 10 min at 3300 g (e.g. 4,000 rpm in tabletop centrifuge) at 4 °C.
5. Discard the medium and resuspend the cell pellet in 30-40 ml cold 0.1M CaCl2.
6. Keep the cells on ice for 30 min.
7. Centrifuge the cells as above.
8. Remove the supernatant, and resuspend the cell pellet in 6 ml 0.1 M CaCl2 solution plus 15% glycerol.
9. Pipet 0.4-0.5 ml of the cell suspension into sterile 1.5 ml micro-centrifuge tubes.
Freeze these tubes on dry ice and then transfer them to -70 C freezer.
Notes:
1. The transformation efficiency is about 1-5x106/ul DNA when using the competent cells prepared with this method.
Important: all steps after harvesting the cell should be done on ice (or at 4 °C)
2. The frozen competent cells are stable for 6 months, but once a tube is taken from the freezer and thawed, any unused portion should be discarded.
3. After the competent cells are made, the transformation efficiency should be checked by transformation using plasmid DNA of known concentration.
Reference:
*Current Protocols in Molecular Biology (1.8.2)
Chemical Transformation of Competent Cells
Before bacteria can be artificially transformed, they have to be made competent—able to take up DNA. The DNA molecule is hydrophilic (water-soluble) but cell membranes are made of a very hydrophobic lipid bilayer, and therefore artificial transformation is not a process that occurs spontaneously. There are two means of artificial transformation commonly used in labs: electroporation and chemical transformation. During electroporation, short bursts of current are passed through a solution containing bacteria at high voltage. The current makes the cell membrane leaky (porous) for a short time, allowing the cells to take up DNA molecules from the solution. In chemical transformation, bacteria are exposed to solutions which alter their cell membranes enough to make the DNA molecules pass through and into the cell. Chemical transformation procedures sometimes also use a heat shock treatment. During this treatment, cells are heat-shocked, then treated with the DNA and a high concentration of calcium ions. The calcium ions precipitate the DNA on the surface of the cell, where the DNA is forced into the recipient. The actual mechanisms by which these two processes work are not fully understood.
- Equilibrate a water bath to 42°C.
- Warm S.O.C medium to room temperature.
- [Spread X-Gal onto LB agar plates with antibiotic, if desired for blue/white selection.]
- Warm the selective plates in a 37°C incubator for 30 minutes (use one plate for each transformation).
- Centrifuge the vial(s) containing the ligation reaction(s) briefly and place on ice.
- Thaw, on ice, one 50µl vial of competent cells for each ligation/transformation.
- Pipet 1 to 5µl of each ligation reaction directly into the vial of competent cells and mix by tapping gently. Do not mix by pipetting up and down. The remaining ligation mixture(s) can be stored at -20°C.
- Incubate the vial(s) on ice for 30 minutes.
- Incubate for exactly 30 seconds in the 42°C water bath. Do not mix or shake.
- Remove vial(s) from the 42°C bath and place them on ice.
- Add 250µl of pre-warmed S.O.C medium to each vial. S.O.C is a rich medium; sterile technique must be practiced to avoid contamination.
- Place the vial(s) in a microcentrifuge rack on its side and secure with tape to avoid loss of the vial(s). Shake the vial(s) at 37°C for exactly 1 hour at 225 rpm in a shaking incubator.
- Spread 20 µl to 200µl from each transformation vial on separate, labeled LB agar plates. The remaining transformation mix may be stored at +4°C and plated out the next day, if desired.
- Invert the plate(s) and incubate at 37°C overnight.
- Select colonies and analyze by plasmid isolation, PCR, or sequencing.
LB/Ampicillin or Kanamycin Cultures
Q: How much ampicillin or kanamycin do we typically use for E.coli?
A: Typically 50 µg/ml which we typically dilute in LB from a 1000X stock ampicillin or kanamycin solution
A: Typically 50 µg/ml which we typically dilute in LB from a 1000X stock ampicillin or kanamycin solution
How To Make Bacterial Glycerol Stabs
Add 0.5ml of the o/n culture to 0.5ml of 70-80% sterile glycerol (in ddH2O) in the sterile screw cap microcentrifuge tube
Pelleting Bacterial Cells
Q: At what rpm should I spin down bacterial (e.g. DH10a E.coli) cells in order to pellet them?
A: ~3000rpm and the time depends on the volume e.g. 3' for 1.5ml or 15' for 50ml
A: ~3000rpm and the time depends on the volume e.g. 3' for 1.5ml or 15' for 50ml
DNA Preps: Recipes for P1, P2, P3 Buffers
Buffer Recipes [from Clontech Adeno-X Mammalian Expression System 1]
P1 (500ml): 1.5gr TRIS pH8, 1.5gr EDTA, 4.5gr Glucose
P2 (500ml): 4gr NaOH, 5gr SDS
P3 (100ml): 49gr KOAc
P1 (500ml): 1.5gr TRIS pH8, 1.5gr EDTA, 4.5gr Glucose
P2 (500ml): 4gr NaOH, 5gr SDS
P3 (100ml): 49gr KOAc
DNA Quantitation By Spectrophotometry
Measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm and 280nm is used as a measure of DNA purity.
DNA absorbs UV light at 260 and 280 nm, and aromatic proteins absorbs UV light at 280 nm; a pure sample of DNA has the 260/280 ratio at 1.8 and is relatively free from protein contamination.
A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1.8.
DNA absorbs UV light at 260 and 280 nm, and aromatic proteins absorbs UV light at 280 nm; a pure sample of DNA has the 260/280 ratio at 1.8 and is relatively free from protein contamination.
A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1.8.
Agarose Gel DNA Electrophoresis
Percent Agarose and Resolution Limits
Q: How much DNA can you see on an agarose gel?
A: By running DNA through an EtBr-treated gel and visualizing it with UV light, any band containing more than ~20ng DNA becomes distinctly visible
Q: What should the ethidium bromide concentration in the gel (or, alternatively, the TAE buffer), be?
A: 0.5 ug/ml
Agarose gel electrophoresis can be used for the separation of DNA fragments ranging from 50 base pair to several megabases (millions of bases) using specialized apparatus. The distance between DNA bands of a given length is determined by the percent agarose in the gel. In general lower concentrations of agarose are better for larger molecules because they result in greater separation between bands that are close in size. The disadvantage of higher concentrations is the long run times (sometimes days). Instead high percentage
agarose gels should be run with a pulsed field electrophoresis (PFE), or field inversion electrophoresis.
Most agarose gels are made with between 0.7% (good separation or resolution of large 5-10kb DNA fragments) and 2% (good resolution for small 0.2-1kb fragments) agarose dissolved in electrophoresis buffer. Up to 3% can be used
for separating very tiny fragments but a vertical polyacrylamide gel is more appropriate in this case. Low percentage gels are very weak and may break when you try to lift them. High percentage gels are often brittle and do not set
evenly. 1% gels are common for many applications.
Q: How much DNA can you see on an agarose gel?
A: By running DNA through an EtBr-treated gel and visualizing it with UV light, any band containing more than ~20ng DNA becomes distinctly visible
Q: What should the ethidium bromide concentration in the gel (or, alternatively, the TAE buffer), be?
A: 0.5 ug/ml
Agarose gel electrophoresis can be used for the separation of DNA fragments ranging from 50 base pair to several megabases (millions of bases) using specialized apparatus. The distance between DNA bands of a given length is determined by the percent agarose in the gel. In general lower concentrations of agarose are better for larger molecules because they result in greater separation between bands that are close in size. The disadvantage of higher concentrations is the long run times (sometimes days). Instead high percentage
agarose gels should be run with a pulsed field electrophoresis (PFE), or field inversion electrophoresis.
Most agarose gels are made with between 0.7% (good separation or resolution of large 5-10kb DNA fragments) and 2% (good resolution for small 0.2-1kb fragments) agarose dissolved in electrophoresis buffer. Up to 3% can be used
for separating very tiny fragments but a vertical polyacrylamide gel is more appropriate in this case. Low percentage gels are very weak and may break when you try to lift them. High percentage gels are often brittle and do not set
evenly. 1% gels are common for many applications.
DNA Precipitation
100% Ethanol (precipitates DNA)
Notes:
1) Sometimes glycogen is used to promote DNA precipitation. Glycogen is a highly purified polysaccharide derived from oysters. It is an inert carrier, free of host DNA/RNA. The Glycogen is insoluble in ethanol solution; in thepresence of salts it forms a precipitate that traps the target nucleic acids. During centrifugation, a visible pellet is formed, which greatly facilitates handling of the target nucleic acids. The Glycogen quantitatively precipitates nucleic acids from diluted solutions with a higher efficiency than that of tRNA, linear polyacrylamide or sonicated DNA. Glycogen molecules are highly branched structures composed of thousands of glucose molecules bonded to each other. The molecular weight of the largest individual glycogen molecule containing about 50,000 glucose molecules appears to be 8 million.
2) Ammonium acetate can be used instead of sodium acetate at 2.0-2.5 M. For example, we can make a 5 M ammonium acetate solution and use a volume equal to that of our sample and then proceed as described above
- add 0.1 volume 3 M sodium acetate
- add 2.5 volumes 100 % Ethanol
- vortex
- precipitate at:
- -20°C overnight (+++)
- -80°C 1 h (++) (preferred)
- dry ice 15min (+)
- spin 20 minutes at 12000 rpm 4°C
- carefully pour out / aspirate supernatant (do not lose DNA-pellet)
70% Ethanol (washes out salt)
- carefully add 1 mL cold 70% Ethanol (do not vortex)
- spin 10 minutes at 12000 rpm 4°C
- carefully pour out / aspirate supernatant (do not lose DNA-pellet)
- air dry 10 minutes at room temperature (do not overdry, because DNA becomes hard to dissolve)
- dissolve in:
- 10 mM Tris pH 7.5 (+++)
- TE-Buffer (++) - EDTA may inhibit downstream enzymatic reactions
- dH2O (+) - freeze at -20°C because unbuffered DNA undergoes degradation
Notes:
1) Sometimes glycogen is used to promote DNA precipitation. Glycogen is a highly purified polysaccharide derived from oysters. It is an inert carrier, free of host DNA/RNA. The Glycogen is insoluble in ethanol solution; in thepresence of salts it forms a precipitate that traps the target nucleic acids. During centrifugation, a visible pellet is formed, which greatly facilitates handling of the target nucleic acids. The Glycogen quantitatively precipitates nucleic acids from diluted solutions with a higher efficiency than that of tRNA, linear polyacrylamide or sonicated DNA. Glycogen molecules are highly branched structures composed of thousands of glucose molecules bonded to each other. The molecular weight of the largest individual glycogen molecule containing about 50,000 glucose molecules appears to be 8 million.
2) Ammonium acetate can be used instead of sodium acetate at 2.0-2.5 M. For example, we can make a 5 M ammonium acetate solution and use a volume equal to that of our sample and then proceed as described above
