Biomedical Engineering Reference
In-Depth Information
ADH1(T1) and CYC1(T2) corresponding to 1,750 bp [
25
].
Fragment 3 was from pRS415 containing the CEN6ARSH4
low copy yeast origin (Fig.
3
).
10. The PHUSER website is capable of including linkers in junc-
tions between DNA fragments. To do so merely include the
desired linker sequence in FASTA format between the two
fragments where the linker is needed and name it “>linker.”
11. Remember that your fi nished plasmids must contain origin and
a selection marker when performing UCF-USER fusion.
12. The PCR program described is optimized for the proof-reading
X-7 polymerase [
30
]. When using the commercial
PfuTurbo
®
Cx Hotstart DNA Polymerase (Agilent) follow the manufac-
turer's instructions.
13. DpnI is a 4 bp restriction enzyme acting on methylated target
DNA. As plasmid DNA isolated from bacteria is methylated,
residual plasmid DNA can be removed by DpnI digestion leav-
ing only non-methylated PCR product. DpnI is functional in
the buffer for proof-reading X-7 polymerase and the buffer for
the
PfuTurbo
®
Cx Hotstart DNA Polymerase.
14. Place the 1 L Blue cap fl ask on the magnetic stirrer and pour in
500 mL 1× TAE buffer along with a Tefl on
®
-coated magnetic
stir bar. Add 10 g SeaKem
®
LE agarose while stirring. Remove
the lid and microwave the fl ask at max effect until the gel mate-
rial is dissolved. Fill the bottle to the 1 L mark with 1× TAE
buffer. Caution: The liquid can become superheated and fl ash
boil upon agitation. The dissolved gel can be stored at 60 °C
for a couple of days.
15. When constructing pBOSAL-2, the DNA fragments 1 and 3
were contaminated with by-products from the PCR. To save
time in verifi cation of correct clone in subsequent steps, it was
chosen to perform a gel purifi cation to remove these impurities
before proceeding. This step is rarely necessary after DpnI
treatment and can often be disregarded.
16. PCR products have a tendency to be very concentrated which
can inhibit the USER reaction. Try adding approximately
50-100 ng/kb per PCR product and fi ll with water to 9
L.
17. Do not perform transformation by electroporation, as the elec-
troshock causes the hybridized tails to dissociate thus ruining
the circular product.
18. If you remove the supernatant by pouring, the residual liquid
is suffi cient for resuspension.
19. Ideally, the negative control plate should not have any colo-
nies. However, even in instances where the colonies on the
μ
