DNA Cloning
Cloning Inserts into Plasmids
DNA Cloning Protocol
To clone you need the following:
Vector DNA or your plasmid construct
Vector DNA can be:
Vector or Plasmid DNA (which needs to be
To clone you need at least several micrograms (3-5 or more) of your
vector DNA.
Insert DNA (DNA you want to insert into the plasmid or vector)
Insert DNA can be:
Oligonucleotide Insert (oligos with restriction cut half-sites annealed and
phosphorylated with PNK)
Restriction Enzyme Generated Insert
PCR Insert (PCR with restriction enzyme cut sites (the whole site is
needed!) in the 5’ end of the primer)
If the Insert you want to clone is less than 60 bp, you can order
oligonucleotides and anneal them together.
If your insert DNA size is small (less than 1000 bp), you do not
theoretically need as much DNA as your vector. If your insert DNA size is
tiny (10-200 bp), you need very little of this DNA theoretically.
In general you should have at least 1 microgram of starting insert DNA.
Preparing Vector DNA for Cloning : Mini-prep, Midi-prep, Maxi-prep
We found that the easiest way to prepare DNA is using a mini-prep kit
(Qiagen). We use several columns for the same vector DNA construct.
For each column we add about 4 mL of LB grown bacteria (we add 2
mL of LB bacteria broth to a 2 mL eppendorf tube and spin twice = 2 X 2
mL = 4 mL)
Tip: Always grow about 5 mL of LB in 15 mL Falcon Tubes, keep the 1
mL as a glycerol bacteria stock frozen in the – 80 C freezer.
How to make a Glycerol Bacterial Stock for the – 80 C Freezer which
will keep for Years (you will never have to streak plates or make
competent cells again! – saves you time)
Simply add 50% glycerol to your bacterial growth LB. This does not
have to be exact ie if you are left with about 1 mL of bacterial LB add
about 500 uL of 100% glycerol and freeze in the – 80 C.
After eluting each column with about 50 uL of EB (elution buffer,
Qiagen, Tris buffer – does NOT contain EDTA), we pool the eluates such
that we have around 200 uL to 400 uL (or 4 – 8 columns per construct or
vector). This is gives enough starting vector DNA for cloning.
Note: we found that the EDTA in TE inhibits subsequent restriction
digestion of the eluted DNA from mini-prep and other plasmid purification
kits.
Preparing Insert DNA for Cloning
Insert DNA must be
Restriction Enzyme Digestion and Cutting Your Plasmid Vector for
DNA Cloning
Plasmid vector is usually cut.
Ligation of Insert and Vector
To Ligate Insert to Vector, use 200 ng of Vector as a guide (people
use as little as 50 ng – N.E.B manual).
Insert ng
Insert size
=
Vector ng
Vector size
Therefore to ligate a 500 bp insert into a 5000 bp vector you would
need
Insert ng
=
Insert ng
=
200 ng X 500
5000
20 ng of insert needed for a 1 : 1 ligation
For a insert 3 : 1 vector ligation you would need: 60 ng
Specify: Vector size:
Specify: Insert size:
bp
bp
Ratio:
For
(moles of insert DNA per mole of vector DNA)
100
ng of vector DNA, add
ng of insert DNA.
Ligation
X uL DNA vector 200 ng
Y uL Insert (calculated ng)
2 uL 10 X Buffer
1 uL T4 DNA Ligase NEB
to 20 uL H20
-------20 uL Total
Take 5 uL of Ligation and add to 100 uL of DH 5 alpha cells Invitrogen
(Subcloning efficiency ok, One Shot better, Max Efficiency best – for hard
ligations but expensive).
Heat Shock
Add 250 uL of SOC to competent Cells
Plate ENTIRE amount on plate.
Grow overnite at 37 C