Constitution of Reagent

Antibiotics

Store:at -20 |C|

Antibiotics

	Store	With	Final
Ampicillin	100 |mg/ml|	|ddH2O|	100 |ug/ml|
Bialaphos	5-10 |mg/ml|	|ddH2O|	5-10 |ug/ml|
Carbenicillin	100 |mg/ml|	|ddH2O|	100 |ug/ml|
Chloramphenicol	34 |mg/ml|	|EtOH|	34 |ug/ml|
Kanamycin	50 |mg/ml|	|ddH2O|	50 |ug/ml|
Tetracycline	15 |mg/ml|		15 |ug/ml|

Buffer or Regents

0.5 |M| |EDTA|

Store:at 4 |C|

A polyamino carboxylic acid and a colourless, water-soluble solid. Its conjugate base is named ethylenediaminetetraacetate. It is widely used to dissolve limescale. Its usefulness arises because of its role as a hexadentate (“six-toothed”) ligand and chelating agent, i.e. its ability to “sequester” metal ions such as Ca2+ and Fe3+. After being bound by EDTA, metal ions remain in solution but exhibit diminished reactivity. EDTA is produced as several salts, notably disodium EDTA and calcium disodium EDTA.

Composition

	required	Final
EDTA 2Na|DOT| 2|H2O|	93.1 |g|	0.5 |M|
NaOH	adjust pH to 8.0
|ddH2O|	up to 500	ml|

Note

|EDTA| does not disolve unless the solution pH goes near by 8.0

|TE| buffer

Store:at RT

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 that chelates cations like Mg2+. The purpose of TE buffer is to solubilize DNA or RNA, while protecting it from degradation.

Composition

	volume	final
1 |M| Tris-HCl (pH 8.0)	1 |ml|	10 |mM|
0.5 |M| EDTA	0.2 |ml|	1 |mM|
|ddH2O|	up to 100 |ml|

After the all reagents has mixed, the buffer should be autoclaved at 121 |C| to ensure sterility.

|PBS| buffer

Store:at RT

Phosphate buffered saline (abbreviated PBS) is a buffer solution commonly used in biological research. It is a water-based salt solution containing sodium chloride, sodium phosphate, and, in some formulations, potassium chloride and potassium phosphate. The buffer’s phosphate groups help to maintain a constant pH. The osmolarity and ion concentrations of the solution usually match those of the human body (isotonic).

Composition

	Formular	required	final
Sodium chloride	NaCl	8.0 |g|	137 |mM|
Disodium hydrogen phosphate	|Na2HPO4|	1.44 |g|	10 |mM|
Potassium chloride	KCl	0.2 |g|	2.7 |mM|
Monopotassium phosphate	|KH2PO4|	0.24 |g|	2.0 |mM|
Hydrochloric acid	HCl	adjust pH 7.4
	|ddH2O|	up to 1 |l|

After the all reagents has mixed, the buffer should be autoclaved at 121 |C| to ensure sterility.

Tris-HCl buffer

Store:at 4 |C|

Composition

	0.5 |M|	1.0 |M|	1.5 |M|	5.0 |M|
Tris	60.55 |g|	121.1 |g|	181.65 |g|	605.5 |g|
|ddH2O|	800 |ml|
0.5 |M| HCl	adjust pH at |RT|
|ddH2O|	up to 1000 |ml|

1. Add 800 |ml| of |ddH2O| and add appropriate mass of tris while mixing solution with stirrer

2. Adjust pH with 0.5 |M| HCl. It may increase the solution temperature because of exothermic reaction

3. Cool the solution to |RT|. pH of Tris solution depend on the temperature thus this step is required.

   Note

   Especially, pH is really important on Electrophoresis so DO NOT SKIP this step

   Note

   pH of Tris solution decrease 0.03 with temperature increment 1 |C| Thus the formular below can be used to estimate the correct pH

   $$\begin{array}{c}p{H}_{correct}=p{H}_{required}-(Temperature-20)\times 0.03\end{array}$$

4. Re adjust pH with 0.5 |M| HCl

5. Add |ddH2O| up to 1000 |ml| and autoclave to ensure sterility

Phosphate buffer

REFERENCE:http://biotech.about.com/od/buffersandmedia/ht/phosphatebuffer.htm Store:at 4 |C|

1. Use the Henderson-Hasselbalch equation (below) to determine what ratio of acid to base is required to make a buffer of the desired pH. Use the pKa value (2.16, 7.21 or 12.32) nearest your desired pH and the ratio will refer to the acid-base conjugate pair that correspond to that pKa.

   $$\begin{array}{cc}pH& =pKa+log\left(\frac{\left[Base\right]}{\left[Acid\right]}\right)\end{array}$$

   Example: pH 8.0

   $$\begin{array}{cc}8.0& =7.21+log\left(\frac{\left[Base\right]}{\left[Acid\right]}\right)\\ log\left(\frac{\left[Base\right]}{\left[Acid\right]}\right)& =8.0-7.21\\ \frac{\left[Base\right]}{\left[Acid\right]}& =10^{0.79}\\ & =6.166\end{array}$$

2. The desired molarity of the buffer is the sum of $\left[Acid\right]+\left[Base\right]$. Calculate required molarity of each

   Example: 1 |M|

   $$\begin{array}{cc}\left[Acid\right]+\left[Base\right]& =1\\ \left[Base\right]& =1-\left[Acid\right]\\ \frac{\left[Base\right]}{\left[Acid\right]}& =6.166\\ \frac{1-\left[Acid\right]}{\left[Acid\right]}& =6.166\\ \left[Acid\right]& =\frac{1}{6.166+1}\\ & =0.14\\ \left[Base\right]& =1-0.14\\ & =0.86\end{array}$$

3. Add appropriate amount of Monosodium phosphate (119.98 |g/mol|) and Disodium phosphate (141.96 |g/mol|)

   Example:

   $$\begin{array}{cc}{x}_{mp}& =0.14\times 119.98\\ & =16.8\left[g\right]\\ y_{dp}& =0.86\times 141.96\\ & =122.1\left[g\right]\end{array}$$

4. Check pH and adjust pH slightly as necessary, using phosphoric acid or sodium hydroxide

5. Once the desired pH is reached, bring the volume of buffer to 1 |l|

100 |mM| |IPTG|

Store:at -20 |C|

1. Dilute 2.38 |g| of |IPTG| to 100 |ml| of |ddH2O|
2. Filter sterize to ensure sterility the solution
3. Store at -20 |C|

Media

|LB| medium

Store:at 4 |C|

A nutritionally rich medium which is primarily used for the growth of bacteria. |LB| media formulations have been an industry standard for the cultivation of |E.coli| as far back as the 1950s. These media have been widely used in molecular microbiology applications for the preparation of plasmid DNA and recombinant proteins. It continues to be one of the most common media used for maintaining and cultivating laboratory recombinant strains of |E.coli|. For physiological studies however, the use of |LB| medium is to be discouraged.

Composition

	Required	Final
Bacto-tryptone	10 |g|	1% w/v
Yeast-extract	5 |g|	0.5% w/v
NaCl	10 |g|	1% w/v
10 |M| NaOH [1]	100 |ul|
|H2O|	up to 1 |l|

[1]	Dissolve 40 |g| of NaOH in 100 |ml| of |ddH2O|

After the all reagents has mixed, the buffer should be autoclaved at 121 |C| to ensure sterility.

|SOB| and |SOC| medium

Store:at 4 |C|

|SOB| is a nutrient-rich bacterial growth medium used for microbiological culture, generally of |E.coli|. It was developed by Douglas Hanahan in 1983 and is an adjusted version of the commonly used |LB| media. Growth of |E.coli| in |SOB| or |SOC| medium results in higher transformation efficiencies of plasmids.

|SOC| is |SOB| with added glucose.

Composition of SOB

	Required	Final
Bacto-tryptone	20 |g|	2% w/v
Yeast-extract	5 |g|	0.5% w/v
10 |mM| NaCl	0.584 |g|
2.5 |mM| KCl	0.186 |g|
|H2O|	up to 1 |l|

Composition of SOC

	Required	Final
Bacto-tryptone	20 |g|	2% w/v
Yeast-extract	5 |g|	0.5% w/v
10 |mM| NaCl	0.584 |g|
2.5 |mM| KCl	0.186 |g|
10 |mM| |MgCl2|	0.952 |g|
20 |mM| glucose	3.603 |g|
|H2O|	up to 1 |l|

After you mix the reagents, adjust pH to 7.0 by adding NaOH for maximum effectiveness. Finally the |SOB| medium should be autoclaved at 121 |C| to ensure sterility. |SOC| medium should not be autoclaved because the high temperature can cause the glucose to react with tryptic peptides. |SOC| can instead be filter sterilized through a 0.22 |um| filter. Alternatively, |SOB| and magnesium and glucose additive solutions can be autoclaved separately and mixed afterwords to final concentrations, although any autoclaving may cause glucose to undergo caramelization.

M9ZB medium

Store:at 4 |C|

M9ZB medium is the combination of M9 [2] and ZB [3]

Composition of Pre-M9ZB

Name	Formular	Required	Final
Bacto-tryptone		10 |g|	1% w/v
Disodium hydrogen phosphate	|Na2HPO4|	15 |g|	1.5% w/v
Monopotassium phosphate	|KH2PO4|	3 |g|
Sodium chloride	NaCl	0.5 |g|
Ammonium chloride	|NH4Cl|	1 |g|
Diluted water	|dH2O|	up to 1 |l|

After the all reagents has mixed, the medium should be autoclaved at 121 |C| to ensure sterility. Finally add the following reagents before use.

• Autoclaved $\frac{1}{100}$ volume of 20% glucose (store at 4 |C|)
• Autoclaved $\frac{1}{500}$ volume of 1 |M| |MgSO4| (store at 4 |C|)
• Autoclaved $\frac{1}{10000}$ volume of 1 |M| |CaCl2| (store at 4 |C|)

[2]	M9 medium is a minimal growth medium used for bacterial cultures. It has the advantage of being cheap and has a very low autofluorenscence and also very low absorbance.

[3]	ZB is a specially formulated growth medium used for the preparation of highly competent |E.coli| cells for DNA transformation.

Note

To create 100 |ml| of M9ZB ready to use, follow the instruction below

1. Transfer 100 |ml| of Pre-M9ZB to 100 |ml| medium bottle and autoclave at 121 |C| to ensure sterility. Autoclave 20% glucose, 1 |M| |MgSO4| and 1 |M| |CaCl2| as well if you don’t have autoclaved one.

2. Add the followings to 100 |ml| medium aspetically. I dispense each reagents to dry heat sterilized 10 |ml| beaker and transfer appropriate volume of each reagents with pipet.

   	Volume	Final
   20% glucose	1 |ml|	0.2%
   1 |M| |MgSO4|	200 |ul|	2 |mM|
   1 |M| |CaCl2|	10 |ul|	10 |mM|

3. Add appropriate antibiotics to the medium if needed and store at 4 |C|

|LB| Agar plate

This protocol assumed to create 40 |LB| Agar plates.

Procedure

1. Weight out the following reagents and put to 500 |ml| of a graduated cylinder

   • 5.0 |g| of Bacto-tryptone
   • 2.5 |g| of Yeast-extract
   • 5.0 |g| of NaCl
   • 7.5 |g| of Agar

2. Add approximately 100 |ml| of |dH2O| and mix powder well to bring into solution

3. Add |dH2O| to total volume of 500 |ml| and transfer to 1 |l| flask

4. Put on stirring plate and stirring for mixing well

5. Autoclave at liquid setting for 20 min in a basin making sure to loosen top

   Note

   Label with autoclave tape is recommended in case

6. Let agar cool to approximately 55 |C| (you should be able to pick up the jar without a glove)

7. Make sure bench top has wiped down with 100% |EtOH|

8. Remove sterile petri dishes from plastic bag and pour a thin layer (5 |mm|) of LB Agar (~10 |ml|) into each plate being careful to not lift the cover off excessively

   Note

   If you need a plate with antibiotic(s), add appropriate volume (0.5 |ml| for stocked antibiotics in Antibiotics concentration ) is of antibiotic(s) just before pouring to dishes.

9. Swirl plate in a circular motion to distribute agar on bottom completely

10. Let each plate cool until its solid (~20 min) then flip so as to avoid condensation on the agar

11. Store plates in plastic bags in fridge with: name, date and contents (note any additive)

SDS-PAGE

3x |SDS| Sample buffer

REFERENCE:http://cshprotocols.cshlp.org/content/2006/1/pdb.rec10075.full?text_only=true Store:at |RT|

Composition

	Required	Final
1 |M| Tris-HCl (pH 6.8, autoclaved)	1.88 |ml|	188 |mM|
Glycerol	3 |ml|	30%
10% |SDS|	3 |ml|	3%
|BPB|	0.01 |g|	0.01%
|ddH2O|	up to 10 |ml|

After you mixed the reagents, filtrate the solution with 0.2 |um| filter and add |b-ME| to 15% of the solution before use

6x |SDS| Sample buffer

REFERENCE:http://cshprotocols.cshlp.org/content/2006/1/pdb.rec10075.full?text_only=true Store:at |RT| (without |b-ME|) or at 4 |C| for several month (with |b-ME|)

Composition

	Required	Final
5 |M| Tris-HCl (pH 6.8, autoclaved)	0.752 |ml|	376 |mM|
Glycerol	6 |ml|	60%
|SDS|	0.6 |g|	6%
|BPB|	0.02 |g|	0.02%
|ddH2O|	up to 7 |ml|

Filtrate the solution with 0.2 |um| filter and dispense 700 |ul| to eppendolf tubes. Add 300 |ul| of |b-ME| (final of 30%) just before use and store at 4 |C| for several month [4]

[4]	Some said store it in 4 |C| for long time should be OK. http://forums.biotechniques.com/viewtopic.php?f=4&t=779

10x SDS Running buffer

Store:at |RT|

Composition

	Required	Final
Tris-base	30.28 |g|	25 |mM|
Glycine	143.1 |g|	191 |mM|
|SDS|	10 |g|	0.1%
|ddH2O|	up to 1 |l|

30% Acrylamide stock solution

Store:at 4 |C|

Composition

	Required
Acrylamide (monomer)	58 |g|
Bisacrylamide	2 |g|
|ddH2O|	up to 200 |ul|

Conduct filtration (0.4 um) before use

Warning

Acrylamide and solution used acrylamide (before coagulate) has neurotoxin.

10% Ammonium persulfate (APS)

Store:4 |C| for quick store

Composition

Ammonium Persulfate	1 |g|
|ddH2O|	up to 1 |ml|

Note

For SDS Polyacrylamide gel electrophoresis (SDS-PAGE), you can just add 1 |ml| of |ddH2O| to 1 |g| of Ammonium Persulfate because the concentration is unimportant

Note

10% Ammonium persulfate can be store several days at 4 |C| and several weeks at -20 |C|

2x Running Gel buffer

Store:at |RT|

Composition

	required	Final	Final in GEL
1.5M Tris-HCl (pH 8.8)	250 |ml|	0.75 |M|	375 |mM|
10% SDS	20 |ml|	0.8%	0.4%
|ddH2O|	up to 500 |ml|

2x Stacking Gel buffer

Store:at |RT|

Composition

	required	Final	Final in GEL
0.5M Tris-HCl (pH 6.8)	250 |ml|	0.25 |M|	125 |mM|
10% SDS	10 |ml|	0.4%	0.2%
|ddH2O|	up to 500 |ml|

Protein purification

0.2 |M| |PMSF|

Store:at |RT|

Composition

	required	Final
|PMSF|	350 |mg|	35 |mg/ml|
|EtOH|	10 |ml|

Warning

|PMSF| deactivate in |H2O| within 30 min but it is more stable in |EtOH| or isopropyl alcohol. And |PMSF| is injuries thus do not hand it with your hands.

1 |mM| Pepstatin

Store:at -20 |C|

Composition

	required	Final
Pepstatin	6.86 |g|	685 |mg/ml|
Methanol	10 |ml|

1 |mM| Leupeptin

Store:at -20 |C|

Composition

	required	Final
Leupeptin	4.27 |g|	427 |mg/ml|
|ddH2O|	10 |ml|

Extracting buffer (Homogenise solution)

Store:DO NOT STORE

Composition

	required	Final
1 |M| Tris-HCl (pH 7.5 at 25 |C|)	2.5 |ml|	50 |mM|
2 |M| KCl	1 |ml|	40 |mM|
0.5 |M| EDTA (pH 8.0)	0.1 |ml|	1 |mM|
1 |M| |DTT| (at -20 |C|) [5]	0.3 |ml|	1 |mM|
0.2 |PMSF| [6]	1.5 |ml|	1 |mM|
1 |mM| Pepstatin A [7]	0.1 |ml|	2 |uM|
1 |mM| Leupeptin [8]	30 |ul|	0.6 |uM|
|ddH2O|	up to 50 |ml|

[5]	A common small-molecule redox reagent

[6]	A serine protease inhibitor commonly used in the preparation of cell lysates

[7]	A potent inhibitor of aspartyl proteases

[8]	A naturally occurring protease inhibitor that can inhibit cysteine, serine and threonine peptidases

Ni-NTA affinity column

REFERENCE:NOVAGEN Ni-NTA His-Bind Resins Handbook REFERENCE:http://www.pssj.jp/archives/Protocol/Purification/NTA_01/NTA_01_01.html REFERENCE:http://en.wikipedia.org/wiki/Tris REFERENCE:YMZK Tsuda manual

Note

Some protocols use Tris-HCl buffer for buffer solution however tris may intract with proteins thus I use Phosphate buffer insted

Ni-NTA Binding buffer

Store:at 4 |C|

Composition (5x)

	required	Final	Final (1x)
1 |M| Phosphate buffer (pH 8.0) [9]	25 |ml|	250 |mM|	50 |mM|
NaCl	8.77 |g|	1500 |mM|	300 |mM|
Imidazole	0.34 |g|	50 |mM|	10 |mM|
|ddH2O|	up to 100 |ml|

[9]	Because of pKa of His, His couldn’t bind to the column under pH 8.0

Dilute 10 |ml| of 5x Ni-NTA Binding buffer to 40 |ml| of |ddH2O|

Ni-NTA Wash buffer

Store:at 4 |C|

Composition (5x)

	required	Final	Final (1x)
1 |M| Phosphate buffer (pH 8.0)	25 |ml|	250 |mM|	50 |mM|
NaCl	8.77 |g|	1500 |mM|	300 |mM|
Imidazole	0.68 |g|	100 |mM|	20 |mM|
|ddH2O|	up to 100 |ml|

Dilute 10 |ml| of 5x Ni-NTA Wash buffer to 40 |ml| of |ddH2O|

Ni-NTA Elute buffer

Store:at 4 |C|

Composition (5x)

	required	Final	Final (1x)
1 |M| Phosphate buffer (pH 8.0)	25 |ml|	250 |mM|	50 |mM|
NaCl	8.77 |g|	1500 |mM|	300 |mM|
Imidazole	8.51 |g|	1250 |mM|	250 |mM|
|ddH2O|	up to 100 |ml|

Dilute 10 |ml| of 5x Ni-NTA Elute buffer to 40 |ml| of |ddH2O|

Ni-NTA Strip buffer

Store:at 4 |C|

Composition (5x)

	required	Final	Final (1x)
1 |M| Phosphate buffer (pH 8.0)	25 |ml|	250 |mM|	50 |mM|
NaCl	8.77 |g|	1500 |mM|	300 |mM|
Imidazole	27.23 |g|	4000 |mM|	800 |mM|
|ddH2O|	up to 100 |ml|

Dilute 10 |ml| of 5x Ni-NTA Strip buffer to 40 |ml| of |ddH2O|

Ni-NTA Charge buffer

Store:at 4 |C|

Composition (5x)

	Final	Final (1x)
|NiSO4|	400 |mM|	100 |mM|
|ddH2O|	up to 100 |ml|

1. Add appropriate mass of |NiSO4| to a graduated cylinder. Refer the following table to determine the appropriate mass of your |NiSO4|

   	molecular mass	required	Final	Final (1x)
   |NiSO4|	154.75 |g/mol|	6.19 |g|	400 |mM|	100 |mM|
   |NiSO4-6H2O|	262.85 |g/mol|	10.51 |g|	400 |mM|	100 |mM|
   |NiSO4-7H2O|	280.46 |g/mol|	11.22 |g|	400 |mM|	100 |mM|

2. Bring the volume up to 100 |ml|