CLONING
PCR products or DNA fragments can be easily cloned into cloning vectors
for different prepuces.
Here we describe the cloning of a PCR product (insert) into pGEMteasy vector from promega
with T-tails. These vectors are used to study DNA fragment of interest, for example by
sequencing.
1. Ligation of PCR product with A tails in each end into pGEMT vector.
The kit with catalog Nr. A1380 contains enzyme and competent cells for
cloning.
For each ligation mix we combine:
Vector
Insert
Buffer
Enzyme
Amount (µl)
1
pGEM-T Easy 1.2µg
50 ng/µl (Promega)
2X Rapid Ligation Buffer
(Promega)
T4 DNA Ligase
100u 3u/µl (Promega)
dH2O
Total
X (has to be calculated)
5
1
Y (depend on insert)
10
Finding X:
Formula: ((50 ng/µl (vector) x Z bp (insert)) / 3000 bp (vector) ) x (3/1) =
A ng insert needed
Ex: If the size of the insert is 1200 bp = Z
((50 ng/µl x 1200bp) / 3000) x (3/1) = 60ng = A
A / DNA concentration (measured after gel extraction) = X
Finding Y:
The total volume should be 10µl,
10µl – X – vector – buffer - enzyme = Y
Finally the mix will be incubated at 16oC, over night on a PCR machine.
2. Transformation
JM109 Competent Cells >108 cfu/µg (Promega)
LB Medium
Glucose
1. Chill sterile eppendorf tubes on ice.
2. Thaw frozen Competent Cells on ice (10-15 min).
3. Gently mix the thawed Competent Cells by flicking the tube. Transfer 100 µl
competent cells to a pre-chilled tube.
4. Add 3 µl of ligation mix (DNA), place the tube on ice for 10 minutes.
5. Heat-shock the cells for 50 seconds at 42˚C. Do not shake.
6. Immediately place the tube on ice for 2 minutes.
7. Add cold 1 ml LB medium + 10 µl glucose (2M), 5 ul MgCl2 (1M), 5 ul MgSo4 (1M)
and mix gently but well.
8. Incubate for 1 ½ hour at 37˚C, shake the tube many times by hand during the
incubation time if you are not using a shaker.
3. Making agar plates
Pour 25 ml melted LB agar in each plate. Wait 30 minutes for the agar to solidify. Make two
plates for each sample.
To each plate, add the components according to the table:
Ampicillin (100 mg/ml)
X-Gal
(50 mg/ml, Promega)
IPTG
(0.1 M)
30 µl
20 µl
100 µl
Let the components dry for 15 minutes.
Label the plates, and add 150 µl of the transformation mix to a plate. The remaining part of
the transformation mix is then centrifuged for 1 minute. The bacteria will then make a pellet.
Remove the liquid until 100-150 ul medium remain in the tube. Vortex and resuspend the
bacteria. Transfer the concentrated transformation mix to a new plate.
Incubate at 37 ˚C over night.
4. Screening of colonies
Pick 5 white colonies from each plate, and pick one blue colony. The colonies are placed in
50 ml tubes containing 10 ml LB medium and 10 µl ampicillin(100 mg/ml). Incubate at 37 ˚C
with shaking for 8-16 hours.
Number each of the 50 ml tubes. Transfer from one 50 ml tube to a clean eppendorf tube, 100
µl of the sample. To the eppendorf tube, add 50 µl phenol-chloroform (50% each) and 3 µl
loading buffer (ul volume are depending on the concentration of the loadings buffer). The
same is done with all of the samples in the 50 ml tubes. Vortex all the samples, and centrifuge
them at 150.000 rpm for 5 minutes at room temperature. The samples will now be in two
phases. Load 18 µl from the upper phase to a well in an agarose gel to check if the
ligation/transformation has been successful. The comparative size of the vector in blue and
with colonies will indicate for if the insertion on vector has been successful. Finally the
desired colonies are chosen and plasmid extraction step are conducted.
5. Sequencing
Extracted plasmids with insert will be delivered for sequencing at CEES, University of Oslo.
Sample Delivery
Sequencing
Samples should be delivered in PCR 8-strip tubes for easier handling. All
samples should contain a mix of both template and primer.
Sample quality is essential. Automated DNA sequencing requires a higher
degree of template purity than manual sequencing. Inadequate DNA clean-up is
the most common cause of poor sequencing results. The ABI-lab keep a supply of
Exozap-IT for sale, please contact the staff.
Sequencing is currently done twice a week, Monday and Wednesday.
Samples delivered before 12 noon on Monday or Wednesday will be sequenced
within approximately 48 hours, unless sample amount exceedes capacity or
unforseen events prohibits sequencing.
-Sample delivery takes place in room 3424.
The samples are placed in the fridge at room 3424, and the paper version of the
delivery form is placed in the adjacent box.
Sequencing of plasmids:
For sequencing of plasmids we require 8 µl of template with an approximate
concentration of 20-100 ng/µl mixed with 2µl of a 5 µM primer.
We recommend that new users deliver no less than 250ng total amount of
plasmids until we have established a working protocol for you.
Sequencing PCR products:
All PCR products should be cleaned in advance with a suitable method. As long
as you have a reasonably strong band on your gel electrophoresis, a 10X diluted
PCR sample contain sufficient amounts of DNA for sequencing. When
optimizing your PCR reaction, please lower the amount of primer to an absolute
minimum, as this seems to affect sequencing results. In some cases it may be
problematic to sequence successfully with the same primer as in the PCR
reaction.
We require 8 µl of PCR product, with 2 µl of a 5 µM primer added.
* -For strong PCR bands dilute 10X. For weak PCR bands do not dilute.
Fragment analysis
For fragment analysis, please contact the staff abi-samples@bio.uio.no