Transformation of competent cells and clone screening
Written by Steve Doyle (s.doyle@latrobe.edu.au) - March 2014
-
-
-
transformations involve taking intact plasmid DNA, or ligated plasmid/PCR product,
and using bacteria to cultivate them
bacteria take up some of the DNA during the heat-shock process at 42C, when small
pores form in the bacterial membrane
bacteria that do take up a plasmid gain a selective advantage; plasmids contain
antibiotic resistance genes in them, and when spread onto on an agar plate that
contains antibiotics, only those bacteria that contain a plasmid will survive
this results in small colonies of bacteria to form on the plate; each colony contains
many identical bacteria that all derive from a single bacterium that took up the DNA
each colony is considered an independent clone, and within each colony, the DNA
can be considered identical, but between colonies, each is considered potentially
different; we therefore usually screen a few different colonies from each plate to
find one that we want
this protocol demonstrates how to do the transformations, how to screen colonies
for the plasmid and insert (colony PCR), and how to preserve the bacteria containing
the plasmid DNA for future use (glycerol stocks)
Preparation
-
LB broth
-
LB / agar plates containing appropriate antibiotic
-
30% glycerol / LB
-
competent cells
Protocol
Day 1
1. add 2 ul of ligation mix / plasmid DNA (1-10 ng) to a sterile, chilled microcentrifuge
tube
2. transfer 100 ul of competent cells to the tube and swirl with the pipette tip to mix
with the DNA
a.
do not pipette up and down to mix – this will negatively affect that transformation
3. keep tube on ice for 30 mins
4. place tube in a preheated heat block / water bath set to 42°C for exactly 90 seconds
a.
be gentle on do not shake tubes
5. quickly but gentle transfer tubes into ice and allow cells to chill for 1-2 minutes
6. add 500 ul of LB to each tube
7. incubate tubes in a 37°C shaking incubator for 45 minutes
a.
this allows the cells to recover and express the antibiotic resistance gene
8. once removed from the incubator, transfer up to 200 ul onto an agar plate
containing the appropriate antibiotics
a.
ampicillin for pGem-T-Easy cloning
9. spread cells using a sterilized bent glass rod
a.
to sterilize, dip the rod into ethanol and then quickly pass it through the flame of a Bunsen
burner. Do not hold in the flame, but let it burn out by itself. Allow a could of seconds to cool
before spreading.
10. Invert the plates, make sure the plate has been labeled appropriately, and incubate
at 37°C for approximately 16 hours
a.
Do not leave it for too long in the incubator, as non-specific satellite colonies will appear
Day 2
11. Check the plates first thing in the morning. Hopefully they should have some discrete
colonies (white raised spots) growing on the plates. You want the colonies to be
between 1-2 mm wide – no more. If they are too small, put back in the incubators
for another hour or two. When they are ready, take them out of the incubator and
place on the bench.
12. Proceed with either
a. Colony PCR preparation; OR
b. Making overnight cultures
Colony PCR preparation
1. determine how many colonies you want to screen from each plate
a.
if you expect there to be some diversity among clones on the plate, (ie. you are confirming a
heterozygote SNP, or you know you have mixed DNA sequences) pick >5
b.
if you expect the colonies to be essentially the same, pick 3
2. setup one sterile microcentrifuge tube per colony to be screened and into it, aliquot
20 ul of H2O
3. using a P20 pipette and pipette tip, gently touch the end of the tip onto a colony and
transfer it into one of the tubes, swirling and pipetting up and down to mix
a.
you do not need much of the colony to be transferred for this to work; importantly, you do
not want more than one colony being transferred. Avoid colonies that are too close together.
4. Repeat for all colonies to be screened
5. Set up single tubes, 8-well strips or 96-well plates (depending on how many colonies
you have to screen)
6. Add 10 ul of each colony resuspension to individual wells, keeping the remaining 10
ul aside on the bench – DO NOT DISCARD.
7. Place the tube/strip/plate in the PCR machine and incubate a 95°C for 20 minutes to
lyse the bacterial cells
a.
This becomes the template for the PCR
8. Proceed with a relevant PCR amplification, using 1 ul of the bacterial lysate as the
template DNA for the reaction
a.
Primers could be insert specific, plasmid backbone specific (M13, T7), or a combination of
both
9. Once you have confirmed which colonies are positive by PCR and running a gel, use
the remaining 10 ul of bacterial suspension (NOT the lysate) as a starter overnight
culture – proceed with Making Overnight Cultures
Making Overnight Cultures
1. prepare for making overnight cultures but determining how many colonies you are
going screen
2. per colony, aliquot 5 ml of LB broth into a 10 ml tube
3. add sufficient antibiotic to each tube
a.
if using pGem-T-Easy, add 5 ul of 100 ug/ml of ampicillin to each tube
4. using a P20 pipette and pipette tip, gently touch the end of the tip onto a colony and
transfer it into one of the tubes, swirling and pipetting up and down to mix
a.
you do not need much of the colony to be transferred for this to work; importantly, you do
not want more than one colony being transferred. Avoid colonies that are too close together.
b.
If you are proceeding from the colony PCR step, add the entire remaining 10 ul of bacterial
suspension to the tube
5.
Place tubes on roughly a 45 degree angle in an 37C shaking incubator (180 RPM) for
overnight
a.
Placing the tubes on an angle helps aerate the suspension, promoting bacterial growth.
Tubes straight up or lying down do not grow as well.
Day 3
6.
In the morning, check the tubes – they should have changed from clear yesterday to
be cloudy today, which means that the bacteria have successfully grown
7.
For samples that have grown, prepare a glycerol stock of the sample before
proceeding with the miniprep DNA extraction
a.
Glycerol stocks of bacterial cultures can be kept essentially indefinitely in the -80C freezer.
You only really need to make them if you plan to continue to use the DNA you extract time
and time again.
8.
Preparing glycerol stocks
a.
Per sample, add 500 ul of 30% glycerol/LB into a microcentrifuge tube
i. 30% glycerol/LB = 30 ml glycerol + 70 ml LB broth; autoclave to sterilise
9.
b.
Add 500 ul of the bacterial suspension to the 30% glycerol / LB
c.
Place in the -80C freezer
Once glycerol stocks are made, proceed to extract the plasmid DNA using a Miniprep
DNA extraction kit, following the manufacturers instructions
a.
We typically use the entire remaining ~4.5 mls in the Miniprep
10. After you have done your Miniprep, and confirmed your insert is correct, ie. by
Sanger Sequencing, you do not need to keep ALL of the glycerol stocks that you
made in step 8 above – just keep the one that has been confirmed.