Protocols for DNA¶

Agarose gel electrophoresis¶

REFERENCE:	http://openwetware.org/wiki/Agarose_gel_electrophoresis

To separate DNA or RNA molecules by size. Nucleic acids are negatively charged and are moved through an agarose matrix by an electric field. Shorter molecules move faster and migrate further.

Preparation¶

Casting Gels¶

The amount of agarose to use in your gel depends on the DNA in question. Use the following table as a rough guide.

Agarose Concentration	Optimal DNA Resolution
0.5%	1-30 kb
0.7%	0.8-12 kb
1.0%	0.5-10 kb
1.2%	0.4-7 kb
1.5%	0.2-3 kb

Measure out the appropriate mass of agarose into a bottle with the appropriate volume of 0.5% |TBE| buffer

Note

Use 0.8 |g| of Agarose and 80 |ml| of 0.5% TBE buffer for 4 mini 1% gels
Microwave until the agarose if fully melted.

Warning

The solution can explosively boil. So DO NOT TAKE YOUR EYES OFF
Let the agarose cool on your bench until touching the bottom of the bottle with your bare hand doesn’t burn you. The bottle will cool unevenly, so you must be careful not to cause ripples and bubbles.
Pour the agarose solution into the gel-box. Carefully pop or shove to the side any bubbles, put it the comb, and let it cool for about 30 min, until the gel is solid.

Warning

DO NOT PUT COMB DEEP otherwise the gel will be pierced.
Wrap gels with plastic wrap and store at 4 |C| for several weeks

Loading dyes¶

	0.5-1.5% agarose	2.0-3.0% agarose
Xylene cyanol	10,000-4,000 bp	750-200 bp
Cresol Red	2,000-1,000 bp	200-125 bp
Bromophenol blue	500-400 bp	150-50 bp
Orange G	< 100 bp	?
Tartrazine	< 20 bp	< 20 bp

Volume and number of wells¶

The table below show the capacity volume of each type of well and the number of wells in gel.

	Estimated capacity	Mini	Large
Normal well	16 \|ul\|	8	17
Gel extraction well	64 \|ul\|	2+2	2+5
Colony PCR well	8 \|ul\|	13	25

Procedure¶

Set gel on running bath and fill up the bath with 0.5% |TBE| buffer
Add 1 |ul| of |EtBr| and run 30 min to be sure the |EtBr| permeated into the gel

Warning

|EtBr| is thought to act as a mutagen because it intercalates double stranded DNA (i.e., inserts itself between the strands), deforming the DNA. This could affect DNA biological processes, like DNA replication and transcription. |EtBr| has been shown to be mutagenic to bacteria via the Ames test, but only after treatment with liver homogenate, which simulates the metabolic breakdown of the molecule being tested. [1]
Apply the each samples to each well and run 20-40 min

Note

Run 30 min for 1 kb DNA with 1%. Most of time 30 min running is suitable for 0.5-2kb DNA on 1% or 2% gel

[1]	Cite from http://en.wikipedia.org/wiki/Ethidium_bromide#Health_risks

Estimate sample concentration¶

To estimate sample concentration, use ImageJ to find RGB square mean value of marker and each sample bands and background (where no bands appear). And use following formula to calculate estimated mass

Note

Use Alt + M to memorize the informations of selected area. Changing square size of selected area between each bands is not recommended.

System Message: ERROR/3 (/home/docs/sites/readthedocs.org/checkouts/readthedocs.org/user_builds/molecular-biology-protocols/checkouts/latest/DNA.rst, line 126)

Unknown LaTeX command: dots

X               &= G_{marker} \times \frac{V_{sample} - V_{background}}{V_{marker} - V_{background}} \\
\\
X               &\dots \text{An estimated mass of sample in $ng$} \\
G_{marker}      &\dots \text{A mass of marker band in $ng$} \\
V_{sample}      &\dots \text{A RGB square mean value of sample band} \\
V_{marker}      &\dots \text{A RGB square mean value of marker band} \\
V_{background}  &\dots \text{A RGB square mean value of background (where no bands appear)}\\

After sample mass has estimated, simply divide the value with volume of applied.

PCR Amplification¶

TOYOBO KOD -Plus- Neo¶

Reaction Mix¶

	Volume	Final
10x PCR Buffer for KOD -Plus- Neo	5 \|ul\|	1x
2 \|mM\| dNTPs	5 \|ul\|	0.2 \|mM\|
25 \|mM\| \|MgSO4\|	3 \|ul\|	1.5 \|mM\|
Fwd primer (5 \|uM\|)	3 \|ul\|	0.3 \|uM\|
Rev primer (5 \|uM\|)	3 \|ul\|	0.3 \|uM\|
KOD -Plus- Neo (1 U/\|ul\|)	1 \|ul\|	1 U/50 \|ul\|
Template	X \|ul\|	Genomic DNA: 200 \|ng\| Plasmid DNA: 50 \|ng\| cDNA: 200 \|ng\|
\|ddH2O\|	up to 50 \|ul\|

2 Step Cycle Program¶

Initial Denature: 94 |C|, 2 min
25-45 Cycle
1. Denaturation: 98 |C|, 10 sec
2. Annealing & Extension: 68 |C|, 30 sec/kb

3 Step Cycle Program (for |Tm| < 63)¶

Initial Denature: 94 |C|, 2 min
25-45 Cycle
1. Denaturation: 98 |C|, 10 sec
2. Annealing: |Tm| |C|, 30 sec
3. Extension: 68 |C|, 30 sec/kb

Restriction Digest¶

REFERENCE:	http://openwetware.org/wiki/Engineering_BioBrick_vectors_from_BioBrick_parts/Restriction_digest

Calculation¶

Calculate required volume of DNA¶

If you don’t know the required mass of vector and insert DNA, calculate these first. See Calculation section of DNA ligation for more detail.

This calculation assumed that digested DNA is precipitated to 10 |ul| and 2 |ul| for vector, 3 |ul| for insert (two piece ligation) and 1.5 |ul| of each inserts (three piece ligation) is used to ligate DNA.

Each volume of samples in DNA ligation is assumed as

	Two piece ligation	Three piece ligation
Vector	2 \|ul\|	2 \|ul\|
Insert	3 \|ul\|
Insert1		1.5 \|ul\|
Insert2		1.5 \|ul\|

So required mass of each sample is calculated as (assumed 1.5-fold excess is required)

System Message: ERROR/3 (/home/docs/sites/readthedocs.org/checkouts/readthedocs.org/user_builds/molecular-biology-protocols/checkouts/latest/DNA.rst, line 229)

Unknown LaTeX command: dots

G'_{vector}     &= 1.5 \times \frac{10}{2} \times G_{vector}       \\ &= 7.5 \times G_{vector} \\
G'_{insert}     &= 1.5 \times \frac{10}{3} \times G_{insert}       \\ &= 5 \times G_{insert} \\
G'_{insert1}    &= 1.5 \times \frac{10}{1.5} \times G_{insert1}   \\ &= 10 \times G_{insert1} \\
G'_{insert2}    &= 1.5 \times \frac{10}{1.5} \times G_{insert2}   \\ &= 10 \times G_{insert2} \\
\\
G'_{vector}     &\dots \text{Mass of vector required for Digestion in $ng$} \\
G_{vector}      &\dots \text{Mass of vector required for Ligation in $ng$} \\
G'_{insert}     &\dots \text{Mass of insert required for Digestion in $ng$} \\
G_{insert}      &\dots \text{Mass of insert required for Ligation in $ng$} \\
G'_{insert1}    &\dots \text{Mass of insert1 required for Digestion in $ng$} \\
G_{insert1}     &\dots \text{Mass of insert1 required for Ligation in $ng$} \\
G'_{insert2}    &\dots \text{Mass of insert2 required for Digestion in $ng$} \\
G_{insert2}     &\dots \text{Mass of insert2 required for Ligation in $ng$} \\

Calculate required volume of enzyme¶

All restriction digest enzyme has different activity. The activity of each enzyme is shown as unit. The general unit is defined as

One unit is defined as the amount of enzyme required to digest 1 |ug| of lambda DNA in 1 hour at 37 |C| in a total reaction volume of 50 |ul|. [2]

[2]	Cite from http://www.neb.com/nebecomm/products/productR0101.asp

To calculate unit enzyme activity, calculate molar of substrate DNA with a formula below

Note

If you are in rush, just use 1 |ul| of enzyme for digestion. Most of time volume of enzyme required is lower than 1 |ul|.

System Message: ERROR/3 (/home/docs/sites/readthedocs.org/checkouts/readthedocs.org/user_builds/molecular-biology-protocols/checkouts/latest/DNA.rst, line 259)

Unknown LaTeX command: dots

W_{substrate}   &= L_{substrate} \times A \\
M_{substrate}   &= \frac{1}{W_{substrate}} \times 1 \\
X_{substrate}   &= N_{substrate} \times M_{substrate}\\
\\
W_{substrate}   &\dots \text{Molecular weight of substrate DNA in $g$} \\
L_{substrate}   &\dots \text{The length of substrate DNA in bp} \\
A               &\dots \text{An average molecular weight of a pair of nucleotide in dsDNA (approximately 660)} \\
M_{substrate}   &\dots \text{Molar of substrate DNA in $\mu mol$} \\
X_{substrate}   &\dots \text{Molar of sites for definition in $\mu mol$} \\
N_{substrate}   &\dots \text{The number of sites in substrate DNA} \\

The number of restriction site per substrate DNA ( $N_{s u b s t r a t e}$ in formula) is found on http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/frequency_of_restriction_sites.asp

So the restriction enzyme can cut $X_{s u b s t r a t e}$ |umol| restriction sites per 1 hour at 37 |C|.

Note

The definition is the highest activity of the restriction enzyme. Thus assumed 3-fold excess of enzyme is required to cut the same amount of sites described in definition is recommended.

\begin{matrix} X_{s u b s t r a t e} = \frac{N_{s u b s t r a t e} \times M_{s u b s t r a t e}}{3} \end{matrix}

Next we need to know how many sites in digestion solution. To calculate molar of sites in digestion solution, calculate molar of cleavage DNA with a formula below

System Message: ERROR/3 (/home/docs/sites/readthedocs.org/checkouts/readthedocs.org/user_builds/molecular-biology-protocols/checkouts/latest/DNA.rst, line 285)

Unknown LaTeX command: dots

W_{cleavage}   &= L_{cleavage} \times A \\
M_{cleavage}   &= \frac{G_{cleavage}}{W_{cleavage}} \times 1 \\
X_{cleavage}   &= N_{cleavage} \times M_{cleavage}\\
\\
W_{cleavage}   &\dots \text{Molecular weight of cleavage DNA in $g$} \\
L_{cleavage}   &\dots \text{The length of cleavage DNA in bp} \\
A              &\dots \text{An average molecular weight of a pair of nucleotide in dsDNA (approximately 660)} \\
M_{cleavage}   &\dots \text{Molar of cleavage DNA in $\mu mol$} \\
G_{cleavage}   &\dots \text{Mass of cleavage DNA in $\mu g$} \\
X_{cleavage}   &\dots \text{Molar of sites for digestion solution in $\mu mol$} \\
N_{cleavage}   &\dots \text{The number of sites in cleavage DNA} \\

So there are $X_{c l e a v a g e}$ |umol| restriction sites in digestion solution.

Now we know the number of sites which enzyme can cut in 1 hour at 37 |C| and the number of sites in digestion solution. According to these information, calculate required units for complete digestion for 1 hour with a formula below

System Message: ERROR/3 (/home/docs/sites/readthedocs.org/checkouts/readthedocs.org/user_builds/molecular-biology-protocols/checkouts/latest/DNA.rst, line 303)

Unknown LaTeX command: dots

X               &= \frac{X_{cleavage}}{X_{substrate}} \\
                &= \frac{\frac{N_{cleavage} \times G_{cleavage}}{L_{cleavage} \times A}}{\frac{N_{substrate}}{L_{substrate} \times A}} \\
                &= \frac{N_{cleavage} \times G_{cleavage} \times L_{substrate}}{N_{substrate} \times L_{cleavage}} \\
\\
X               &\dots \text{Units required to complete digest in 1 hour} \\
N_{cleavage}    &\dots \text{The number of sites in cleavage DNA} \\
G_{cleavage}    &\dots \text{Mass of cleavage DNA in $\mu g$} \\
L_{substrate}   &\dots \text{The length of substrate DNA in bp} \\
N_{substrate}   &\dots \text{The number of sites in substrate DNA} \\
L_{cleavage}    &\dots \text{The length of cleavage DNA in bp} \\

Note

Assuming 3-fold excess of enzyme is required to cut the same amount of sites described in definition, multiple three to calculation result like below

System Message: ERROR/3 (/home/docs/sites/readthedocs.org/checkouts/readthedocs.org/user_builds/molecular-biology-protocols/checkouts/latest/DNA.rst, line 319)

Unknown LaTeX command: dots

X               &= 3 \times \frac{N_{cleavage} \times G_{cleavage} \times L_{substrate}}{N_{substrate} \times L_{cleavage}} \\
\\
X               &\dots \text{Units required to complete digest in 1 hour} \\
N_{cleavage}    &\dots \text{The number of sites in cleavage DNA} \\
G_{cleavage}    &\dots \text{Mass of cleavage DNA in $\mu g$} \\
L_{substrate}   &\dots \text{The length of substrate DNA in bp} \\
N_{substrate}   &\dots \text{The number of sites in substrate DNA} \\
L_{cleavage}    &\dots \text{The length of cleavage DNA in bp} \\

Procedure¶

Mix the following reagents in eppendorf tube

	Volume	Final
Appropriate 10x Digestion Buffer	4 \|ul\|	1x
\|ddH2O\| or 0.1% BSA	4 \|ul\|	0.01%
Enzyme 1	X \|ul\|
Enzyme 2 or \|ddH2O\|	Y \|ul\|
DNA	M \|ul\|
\|ddH2O\|	up to 40 \|ul\|

Note

Most of time the enzyme volume required is lower than 1 |ul| so if you are in rush, just add 1 |ul| of enzymes

See the site listed below for appropriate digestion buffer:

Vortex and spin down to be sure the solution well mixed
Incubate tubes at 37 |C| for 1-2 hour. Set incubator 80 |C| while waiting
Incubate tubes at 80 |C| for 20 min

Note

Heat inactivation works on most of enzymes but all. See http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/heat_inactivation.asp if heat inactivatin works on your enzymes
Do Ethanol precipitation to 10 |ul| for removing Digestion buffer for next steps

DNA Ligation¶

REFERENCE:	http://openwetware.org/wiki/DNA_ligation
REFERENCE:	http://catalog.takara-bio.co.jp/product/basic_info.asp?unitid=U100004426

Calculation¶

The best mass of plasmid DNA in transformation is known as 50 |ng| for pUC vector (2,700 bp). With this condition, the best mass of vector is calculated with formula below

System Message: ERROR/3 (/home/docs/sites/readthedocs.org/checkouts/readthedocs.org/user_builds/molecular-biology-protocols/checkouts/latest/DNA.rst, line 388)

Unknown LaTeX command: dots

G_{vector}  &= 50 \times \frac{L_{plasmid}}{2700} \\
\\
G_{vector}  &\dots \text{Required mass of vector in $ng$} \\
L_{plasmid} &\dots \text{A full length of a plasmid (vector + insert) in bp} \\

The insert to vector molar ratios can have a significant effect on outcome of a ligation and subsequent transformation step. The insert to vector molar ratios can vary from a 1:1 to 10:1. A general best insert to vector molar ratio is known as 6:1. With this condition, the best mass of insert is calculated with formula below

System Message: ERROR/3 (/home/docs/sites/readthedocs.org/checkouts/readthedocs.org/user_builds/molecular-biology-protocols/checkouts/latest/DNA.rst, line 398)

Unknown LaTeX command: dots

G_{insert}  &= 6 \times G_{vector} \times \frac{L_{insert}}{L_{vector}} \\
\\
G_{insert}  &\dots \text{Required mass of insert in $ng$} \\
L_{vector}  &\dots \text{The length of vector in bp} \\
L_{insert}  &\dots \text{The length of insert in bp} \\

In the step of transformation, I use 1 |ul| of ligated DNA solution (10 |ul|) to transform. That’s mean the volume required in DNA ligation is 10-fold excess. With this condition, the final required mass of vector and insert are calculated with formula below

System Message: ERROR/3 (/home/docs/sites/readthedocs.org/checkouts/readthedocs.org/user_builds/molecular-biology-protocols/checkouts/latest/DNA.rst, line 409)

Unknown LaTeX command: dots

G_{vector}  &= 10 \times 50 \times \frac{L_{vector} + L_{insert}}{2700} \\
            &= 0.185 \times (L_{vector} + L_{insert}) \\
G_{insert}  &= 6 \times G_{vector} \times \frac{L_{insert}}{L_{vector}} \\
\\
G_{vector}  &\dots \text{Required mass of vector in $ng$} \\
G_{insert}  &\dots \text{Required mass of insert in $ng$} \\
L_{vector}  &\dots \text{The length of vector in bp} \\
L_{insert}  &\dots \text{The length of insert in bp} \\

Procedure¶

This procedure assumed mass of each DNA sample has adjusted in Digestion step.

Add 2 |ul| of vector DNA and 3 |ul| of insert DNA for two piece ligation, 1.5 |ul| of each insert DNAs for three piece ligation to PCR tube
Add 5 |ul| of TAKARA DNA Ligation Kit
Incubate 30 min at 16 |C|. PCR is suitable for this incubation

Note

You can incubate 5 min at 25 |C| for simple ligation.
Transform 1 |ul| of ligated DNA to competent cell. See Protocols for |E.coli| for more detail

Ethanol precipitation¶

REFERENCE:	http://openwetware.org/wiki/Ethanol_precipitation_of_nucleic_acids

Procedure¶

Add the following to your sample in the order they appear and mix with vortex mixer
1. 1/10 volume of 3 |M| |NaOAc|, pH 5.2
2. 2.5 volumes of 100% |EtOH|
3. 1/10 volume of glycogen
Spin at 15,000 rpm in a standard microcentrifuge at |RT| for 15 min. Make sure to mark the outermost edge of the tube so you can find the pellet easily.

Note

If you are in rush, you can spin for 10 min but doing so may reduce recover efficiency
Decant (or carefully pipet off) the supernatant and wash pellet with 50 |ul| of 70% |EtOH|. Mix by inverting the tube. DO NOT USE vortex mixer

Note

You don’t have to decant the supernatant completely in this step
Spin at 15,000 rpm in a standard microcentrifuge at |RT| for 10 min. Make sure to mark the outermost edge of the tube so you can find the pellet easily.

Note

If you are in rush, you can spin for 2 min but doing so may reduce recover efficiency
Remove the supernatant completely with carefully pipetting off and dry the pellet. For this you can air dry (tubes open, ~15 min)
Add your desired quanti of water. Mix with vortex mixer and spin down to resuspend.

Note

Using |TE| buffer is recommend. This makes sure your nucleic acid is at a neutral pH and the |EDTA| will chelate any trace metals. Since they are in such small amounts, neither the buffer nor the |EDTA| will affect most downstream reactions.

Sequencing¶

Compose vector and insert DNA¶

This section explain how to compose vector and insert DNA in general way.

Procedure¶

This takes at least four days to extract ligated plasmid DNA.

Prepare samples (several hours)

See PCR Amplification section for detail. After amplification, you have to purify the product DNA because there are polymerase/endonuclease exists and these protein may affect the downstream steps.

If you want to use extracted DNA from plasmid, be sure that the concentration of sample is enough.
Estimate sample concentration (1 hour)

See Agarose gel electrophoresis and Estimate sample concentration section for detail.

Calculation (15 min)

Use the following formula to calculate required mass for Digestion

System Message: ERROR/3 (/home/docs/sites/readthedocs.org/checkouts/readthedocs.org/user_builds/molecular-biology-protocols/checkouts/latest/DNA.rst, line 562)

Unknown LaTeX command: dots

X_{vector}  &= 1.3875 \times L_{plasmid} \\
X_{insert}  &= 4 \times X_{vector} \times \frac{L_{insert}}{L_{vector}}\\
X_{insert1} &= 8 \times X_{vector} \times \frac{L_{insert1}}{L_{vector}}\\
X_{insert2} &= 8 \times X_{vector} \times \frac{L_{insert2}}{L_{vector}} \\
\\
X_{vector}  &\dots \text{Required mass of vector for Digestion in $ng$} \\
X_{insert}  &\dots \text{Required mass of insert for Digestion in $ng$} \\
X_{insert1} &\dots \text{Required mass of insert1 for Digestion in $ng$} \\
X_{insert2} &\dots \text{Required mass of insert2 for Digestion in $ng$} \\
L_{plasmid} &\dots \text{A full length of plasmid DNA (vector + insert) in bp} \\
L_{vector}  &\dots \text{The length of vector DNA in bp} \\
L_{insert}  &\dots \text{The length of insert DNA in bp} \\
L_{insert1} &\dots \text{The length of insert1 DNA in bp} \\
L_{insert2} &\dots \text{The length of insert2 DNA in bp} \\

Digestion (2 hours)

Digest appropriate mass of sample DNA. Adjust the volume of digestion solution with |ddH2O|. See Restriction Digest section for detail. The section assumed the same condition of this procedure.

After digested, precipitate to 10 |ul| by Ethanol precipitation
Ligation (1 hour)

See DNA Ligation section for detail. The section assumed the same condition of this procedure.
Transformation (overnight)

See Protocols for |E.coli| protocols for detail. Incubate at 37 |C| overnight
Colony PCR (several hours)

See Protocols for |E.coli| protocols for detail. Most of time 5 colonies for each sample is enough to pick (depends on the ligation difficulty)
Single Colony Isolation (overnight)

See Protocols for |E.coli| protocols for detail.
Transfer isolated colony to medium (overnight)

To extract plasmid DNA, pick a colony and dilute it to 2 |ml| of SOC and incubate at 37 |C| overnight
Extract plasmid DNA from overnight cultured medium (1 hour)

Use Plasmid extraction Kit to extract plasmid from cultured medium
Sequencing (several hours)

See Sequencing section for detail.

	Volume	Final
10x PCR Buffer for KOD -Plus- Neo	5 \|ul\|	1x
2 \|mM\| dNTPs	5 \|ul\|	0.2 \|mM\|
25 \|mM\| \|MgSO4\|	3 \|ul\|	1.5 \|mM\|
Fwd primer (5 \|uM\|)	3 \|ul\|	0.3 \|uM\|
Rev primer (5 \|uM\|)	3 \|ul\|	0.3 \|uM\|
KOD -Plus- Neo (1 U/\|ul\|)	1 \|ul\|	1 U/50 \|ul\|
Template	X \|ul\|	Genomic DNA: 200 \|ng\| Plasmid DNA: 50 \|ng\| cDNA: 200 \|ng\|
\|ddH2O\|	up to 50 \|ul\|

Protocols for DNA¶

Agarose gel electrophoresis¶

Preparation¶

Casting Gels¶

Loading dyes¶

Volume and number of wells¶

Procedure¶

Estimate sample concentration¶

PCR Amplification¶

TOYOBO KOD -Plus- Neo¶

Reaction Mix¶

2 Step Cycle Program¶

3 Step Cycle Program (for |Tm| < 63)¶

Restriction Digest¶

Calculation¶

Calculate required volume of DNA¶

Calculate required volume of enzyme¶

Procedure¶

DNA Ligation¶

Calculation¶

Procedure¶

Ethanol precipitation¶

Procedure¶

Sequencing¶

Compose vector and insert DNA¶

Procedure¶

Table Of Contents

Previous topic

Next topic

This Page

Navigation

Protocols for DNA¶

Agarose gel electrophoresis¶

Preparation¶

Casting Gels¶

Loading dyes¶

Volume and number of wells¶

Procedure¶

Estimate sample concentration¶

PCR Amplification¶

TOYOBO KOD -Plus- Neo¶

Reaction Mix¶

2 Step Cycle Program¶

3 Step Cycle Program (for |Tm| < 63)¶

Restriction Digest¶

Calculation¶

Calculate required volume of DNA¶

Calculate required volume of enzyme¶

Procedure¶

DNA Ligation¶

Calculation¶

Procedure¶

Ethanol precipitation¶

Procedure¶

Sequencing¶

Compose vector and insert DNA¶

Procedure¶

Table Of Contents

Previous topic

Next topic

This Page

Quick search

Navigation