Restriction Mapping (Online)

1- Introduction to NCBI
2- Retrieve sequence from NCBI
3- NEBCUTTER  restriction map  selection of restriction enzymes
Restriction Digestion
Equipment:
A) Pipettes
B) Incubator
C) Eppendorf
Procedure:
An analytical-scale restriction enzyme digestion is usually performed in a volume of 20μl with 0.2–1.5μg
of substrate DNA and a two- to tenfold excess of enzyme. If an unusually large volume of DNA or
enzyme is used, aberrant results may occur.
 Select restriction enzymes to digest your plasmid.
 Determine an appropriate reaction buffer by reading the instructions for your enzyme.
****TiP: If you are conducting a double digest (digesting with two enzymes at the same time), you will
need to determine the best buffer that works for both of your enzymes. Most companies will have a
compatibility chart, such as the double digest finder tool from NEB.
 In a 1.5mL tube combine the following: DNA/ Restriction Enzyme(s)/Buffer/ BSA (if
recommended by manufacturer)/ dH2O up to total volume
****TiP: The amount of DNA that you cut depends on your application. A diagnostic digest typically
involves ∼500 ng of DNA, while molecular cloning often requires 1 µg of DNA. The total reaction
volume usually varies from 10-50 µL depending on application and is largely determined by the volume
of DNA to be cut.
 A typical restriction digestion reaction could look like this:
A) 1 µg DNA
B) 1 µL of each Restriction Enzyme
C) 3 µL 10x Buffer
D) 3 µL 10x BSA (if recommended)
E) x µL dH2O (to bring total volume to 30µL)
****TiP: The amount of restriction enzyme you use for a given
digestion will depend on the amount of DNA you want to cut. By
definition: one unit of enzyme will cut 1 µg of DNA in a 50 µL
reaction in 1 hour. Using this ratio, you can calculate the minimal
amount of enzyme for your reaction. However, keep in mind that
restriction enzyme activity is determined under ideal conditions
with very clean DNA, so using a little more enzyme is advisable.
Reactions are often performed with 0.2-0.5 µL of enzyme because
it is difficult to pipette less volume than this; 0.2-0.5 µL will likely
be more enzyme than you will need, but that's okay because a
little more enzyme is usually better.
 Mix gently by pipetting.
 Incubate tube at appropriate temperature (usually 37
°C) for 1 hour. Always follow the manufacturer’s
instructions.
****TiP: Depending on the application and the amount of DNA
in the reaction, incubation time can range from 45 mins to overnight. For diagnostic digests, 1-2 hours is
often sufficient. For digests with >1 µg of DNA used for cloning, it is recommended that you digest for at
least 4 hours.
 To visualize the results of your digest, conduct gel electrophoresis.
 Reference: https://www.addgene.org/protocols/restriction-digest/

Protocol for DNA Digestion with a single restriction enzyme

 Add components to a clean tube in the order shown:
1) 1 µL DNA (concentration 1 µg/µL)
2) 2 µL 10x buffer (available with enzymes)
3) 1 µL restriction enzyme
4) 16 µL sterile water
 Incubate the reaction at digestion temperature (usually 37°C) for 1 hour.
 Stop the digestion by heat inactivation (65°C for 15 minutes) or addition of 10mM final
concentration EDTA.
5) The digested DNA is ready for use in research applications.
Protocol for DNA Digestion with two restriction enzymes
 Add components to a clean tube in the order shown
1) 1 µL DNA (concentration 1 µg/µL)
2) 2 µL 10x buffer
3) 1 µL each restriction enzyme
4) 15 µL sterile water
 Incubate the reaction at digestion temperature (usually 37°C) for 1 hour.
 Stop the digestion by heat inactivation (65°C for 15 minutes) or addition of 10mM final
concentration EDTA.
 The digested DNA is ready for use in research applications.
Reference: (Restriction digestion)
https://www.sigmaaldrich.com/technical-documents/protocols/biology/restriction-
enzyme-digest.html
DNA Ligation
Background Information
 The final step in the construction of a recombinant plasmid is connecting the insert DNA (gene or
fragment of interest) into a compatibly digested vector backbone.
 This is accomplished by covalently connecting the sugar backbone of the two DNA fragments. This
reaction, called ligation, is performed by the T4 DNA ligase enzyme.
 The DNA ligase catalyzes the formation of covalent phosphodiester linkages, which
permanently join the nucleotides together. After ligation, the insert DNA is physically attached to
the backbone and the complete plasmid can be transformed into bacterial cells for propagation.
 The majority of ligation reactions involve DNA fragments that have been generated by restriction
enzyme digestion. Most restriction enzymes digest DNA asymmetrically across their recognition
sequence, which results in a single stranded overhang on the digested end of the DNA fragment.
 The overhangs, called "sticky ends", are what allow the vector and insert to bind to each other.
When the sticky ends are compatible, meaning that the overhanging base pairs on the vector and
insert are complementary, the two pieces of DNA connect and ultimately are fused by the ligation
reaction.

 The example below depicts the ligation of two sticky ends that were generated by EcoRI
digestion:

Usually, scientists select two different enzymes for adding an insert into a vector (one enzyme on the 5'
end and a different enzyme on the 3' end). This ensures that the insert will be added in the correct
orientation and prevents the vector from ligating to itself during the ligation process. If the sticky ends
on either side of the vector are compatible with each other, the vector is much more likely to ligate to
itself rather than to the desired insert. If you are in this situation, it is important to treat the digested
vector backbone with a phosphatase before performing the ligation reaction (phosphatase removes the
5' phosphate and therefore prevents the ligase from being able to fuse the two ends of the vector
together).

 Before setting up the ligation reaction itself, it is important to determine the amount of cut insert
and vector to use for the ligation reaction.
 The volume of vector DNA and insert DNA used in the ligation will vary depending on the size of
each and their concentration.
 However, for most standard cloning (where the insert is smaller than the vector) a 3 insert: 1
vector molar ratio will work just fine. We recommend around 100ng of total DNA in a standard
ligation reaction. Use a ligation calculator to easily quantify how much vector and insert DNA to
use.

Combine the following in a PCR or Eppendorf tube:

 Vector DNA
 Insert DNA
 Ligase Buffer (1μL/10μL reaction for 10X buffer, and 2μL/10μL reaction for 5X buffer)
 0.5-1μL T4 DNA Ligase
 H2O to a total of 10μL
 Incubate at room temperature for 2hr, or at 16°C overnight (following the manufacturer’s
instructions).

Note: For many ligation reactions, especially if using "high concentration" ligase, 5min at room
temperature is enough.

Note: If the DNA concentrations are low such that you cannot get all 100ng of DNA, buffer and ligase
into a 10μL reaction, scale the reaction size as necessary - being sure to increase the amount of buffer
proportionally. 1μL of ligase should be sufficient for larger ligation reactions.

Note: Because ligase buffer contains ATP, which degrades upon freeze/thaw cycles, it is a good idea to
take a fresh tube, thaw it one time and aliquot individual tubes of 5, 10 or 20μL for storage at -20°C.
Whenever you need to set up ligations in the future you can thaw a new tube that you know has only
been thawed once before.
Reference: DNA ligation
https://www.addgene.org/protocols/dna-ligation/
YouTube Video
https://www.youtube.com/watch?v=Ik_Pxht1LM0

Preparation of Competent cells

Procedure:
 Culture bacterial cells in LB media for overnight
 Transfer cultured bacteria in a separate flask and culture separately for one hour at 37 Celsius (till
the OD600  Reach at 0.3 – o.4).
 Transfer the culture in separate flask and centrifuge for 15mints at 6000rpm by keeping
temperature at 4 Celsius.
 Discard the supernatant and suspend the pellet in 20ml of 50mM of CaCl2 (Ice chilled)  Incubate
it on ice for 45 minutes.
 Centrifuge the mixture for 15mints at 6000rpm.
 Discard the supernatant and resuspended the pellet in 2ml of ice chilled 50mM CaCl2 and store.
Transformation
Introduction
 Transformation is the process by which foreign DNA is introduced into a cell.
 Transformation of bacteria with plasmids is important not only for studies in bacteria but also
because bacteria are used as the means for both storing and replicating plasmids.
 Because of this, nearly all plasmids (even those designed for mammalian cell expression)
carry both a bacterial origin of replication and an antibiotic resistance gene for use as a
selectable marker in bacteria.
 Commercial competent cells range significantly in their transformation efficiency.
 The lowest efficiency cells are fine for transforming plasmid DNA for the
purposes of storage and amplification.
 Higher efficiency cells are more important if you will be transforming with very
small amounts of DNA or if you're multiple plasmids at once.
Equipment:
A. Shaking incubator at 37 °C
B. Stationary incubator at 37 °C
C. Water bath at 42 °C
D. Ice bucket filled with ice
E. Microcentrifuge tubes
F. Sterile spreading device
Reagents:
A. LB agar plate (with appropriate antibiotic)
B. LB media
C. Competent cells
D. DNA you'd like to transform
Procedure:
I. Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins).
II. Remove agar plates (containing the appropriate antibiotic) from storage at 4°C and let warm up
to room temperature and then (optional) incubate in 37°C incubator.
III. Mix 1 - 5 μl of DNA (usually 10 pg - 100 ng) into 20-50 μL of competent cells in a microcentrifuge
or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times.
 Transformation efficiencies will be approximately 10-fold lower for ligation of inserts to
vectors than for an intact control plasmid.
IV. Incubate the competent cell/DNA mixture on ice for 20-30 mins.
V. Heat shocks each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C
water bath for 30-60 secs (45 secs is usually ideal, but this varies depending on the competent
cells you are using).
VI. Put the tubes back on ice for 2 min.
VII. Add 250-1,000 μl LB media (without antibiotic) to the bacteria and grow in 37°C shaking
incubator for 45 min.
 This outgrowth step allows the bacteria time to generate the antibiotic resistance
proteins encoded in the plasmid backbone so that they will be able to grow once plated
on the antibiotic containing agar plate. This step is not critical for Ampicillin resistance
but is much more important for other antibiotic resistances
VIII. Plate some or all of the transformation onto a 10 cm LB agar plate containing the appropriate
antibiotic.