Biomedical Engineering Reference
In-Depth Information
•
DNA mini gel
•
Standard submarine gel tank and power pack
•
Hot-start KOD polymerase (Novagen)
•
UV spectrophotometer
•
Pipettes (p2, p10 and p200).
Method
1 Set up 25
μ
l PCR reactions as follows (
μ
l/reaction):
Forward primer (10
μ
M
stock)
a
1.5
μ
l
Reverse primer (10
μ
M
stock)
a
1.5
μ
l
10
×
KOD buffer
2.5
μ
l
dNTPs (provided with KOD)
2.5
μ
l
MgSO
4
(provided with KOD)
1.5
μ
l
Hot-start KOD
0.3
μ
l
∼
100 ng of PCR product
b
1.0
μ
l
d
H
2
O
14.2
μ
l
PCR reactions should be performed using the following program:
i. 98
◦
C for 30 s
ii. 55-68
◦
C for 30 s (dependent on primer
T
m
)
iii. 70
◦
Cfor1min
iv. (extension period should be increased if insert size is larger than 3 Kb)
v. Repeat steps i-iii 29 times
vi. Hold at 15
◦
C.
Run out 5
μ
l of the PCR reaction on a 1% agarose gel to confirm product size and assess
the specificity of the PCR.
c
Notes
a
Primers used to generate inserts for gap repair reactions consist of a standard recombination
tag (A1 or A2), which is identical to the 5
recombination sequence in the vector and a stretch
of approximately 20 3
gene-specific nucleotides. Forward primers may encode the ATG 'start
codon' of the protein of interest, but this is not essential as inserts will be expressed as
in-frame fusions with upstream DB or AD domains. However, it is important to remember that
a 'stop codon' is required at the end of the gene-specific segment when using the pGBAD-B
or pACTBD-B vectors, to facilitate color selection of clones with inserts. Examples of forward
and reverse recombination primers are shown below. Tags to be added to protein or domain of
interest:
