Molecular cloning overview
Steps to prepare vector
1.
12-16 hour
overnight culture
2.
Minipreps
(day immediately
following O/N)
(use Qiaprep spin
miniprep kit)
Measure DNA conc.
3.
Large-scale
restriction enzyme
digest
(Preferably same
day as minipreps)
4.
Large-well agarose
gel
electrophoresis,
ensure bands are
expected sizes, cut
out correct band
from gel
5.
DNA extraction
from agarose
(use Qiaquick gel
extraction kit)
Measure DNA conc.
6.
CIP (calf intestinal
phosphatase)
digestion
7.
DNA precipitation
(with phenol
chloroform, etc)
Measure DNA conc.
Overnight culture may be started from a frozen cell
stock, or colony from agar plate
Start several 5 mL cultures to make sure there is
enough material at the final step (5 or more)
After measuring the DNA concentration to ensure
minipreps were all successful, combine all the DNA into
one microcentrifuge tube for the digest
For pET28b GroES7, the larger band will be at 7500
base pairs
The digest will remove one subunit at 300 base pairs
DNA precipitation procedure takes 2 days
Afterwards, DNA is ready for ligation
Molecular cloning overview
Steps to prepare insert
1.
PCR amplification
2.
Agarose gel,
confirm product is
correct size
3.
TOPO reaction, use
3 uL of PCR
product
4.
Transformation
into supercompetent cells,
plate onto
spectinomycin agar
(prefarably same
day as TOPO
reaction)
5.
12-16 hour
overnight cultures
6.
Minipreps
(day immediately
following O/N)
(use Qiaprep spin
miniprep kit)
Measure DNA conc.
7.
Test digests of
miniprep DNAs
8.
Agarose gel of test
digests, confirm
bands are correct
size
For GroES7 cloning, use GroES single as the template
DNA for PCR, product should be 300 base pairs
Start 4-6 cultures from agar plate
Use 1 uL of DNA if concentration is over 200 ng/uL,
otherwise use 3 uL of DNA, final reaction volume 10 uL
Add 2 uL of 6X DNA loading dye to each sample, load
all 12 uL onto gel
TOPO vector is 2.8 kb
TOPO vector is cut in half by XhoI enzyme, makes one
strong band at 1.4 kb
Molecular cloning overview
Steps to prepare insert, continued
9.
Large-scale digest
of one correct
clone
10.
Large-well agarose
gel
electrophoresis,
ensure bands are
expected sizes, cut
out correct band
from gel
10.
DNA extraction
from agarose
(use Qiaquick gel
extraction kit)
Measure DNA conc.
DNA is ready for ligation
Molecular cloning overview
Ligation
1.
Set up ligation
reactions, let
incubate at room
temperature 8+
hours
2.
Transform 5 uL of
each ligation
reaction into
super-competent
cells
3.
Select 2-6 colonies
from each plate,
set up 12-16 hour
overnight cultures
4.
Minipreps
(day immediately
following O/N)
(use Qiaprep spin
miniprep kit)
Measure DNA conc.
5.
Test digests of
miniprep DNAs
Use 1 uL of DNA if concentration is over 200 ng/uL,
otherwise use 3 uL of DNA, final reaction volume 10 uL
6.
Agarose gel of test
digests, confirm
bands are correct
size
Add 2 uL of 6X DNA loading dye to each sample, load
all 12 uL onto gel
7.
If gel looks good,
set up sequencing
reactions for
correct clones to
confirm
If control plates have many colonies, screen more
clones
Set up and run PCR program 1 before the date of
sequencing plate (not any farther in advance)
Drop off sequencing reactions by 3pm on date of
sequencing plate
Molecular cloning overview
Steps for Quik-Change mutagenesis
(site-directed mutagenesis, point mutation)
1.
PCR amplification
2.
Add 1 uL of DpnI,
digest for 1 hour
3.
Agarose gel,
confirm product is
correct size
4.
Transform into
super-competent
cells,
5.
12-16 hour
overnight cultures
6.
Minipreps
(day immediately
following O/N)
(use Qiaprep spin
miniprep kit)
Measure DNA conc.
7.
Set up sequencing
reactions for
clones to confirm
Do not let digest go for over 1 hour
Start 4-6 cultures from agar plate
Set up and run PCR program 1 before the date of
sequencing plate (not any farther in advance)
Drop off sequencing reactions by 3pm on date of
sequencing plate
Molecular cloning overview
This handbook is a guide- your experiments may be
different, depending on what DNAs are used, and the
final outcome of samples
Last updated 2012 03 19 Melissa Illingworth