Biology Reference
In-Depth Information
4. Review each probe on a 2 % SB-gel. This allows estimation of
the concentration of hybridized oligonucleotides (Fig.
2
).
1. Digest 5-10
g plasmid DNA (pLKO1.puro/pLJM1) with
Age
I and
Eco
RI.
2. Purify digested plasmid DNA using a gel extraction kit and
determine its concentration.
3. Combine 50-100 ng of vector with a three- to tenfold molar
excess (
see
Note 8
) of insert. The amount of insert can be cal-
culated using the following formula:
μ
4.2 Ligation
⎡
⎢
⎤
⎥
×
size insert in bp ng vector
size vector in bp
×
ng insert
=
ratio
4. Plan control reactions: One reaction without insert and one
without enzyme.
Fig. 2
Hybridization effi ciency of oligonucleotides was analyzed by 2 % SB aga-
rose gel electrophoresis. The proportion of single versus double-stranded oligo-
nucleotides can be roughly estimated from the staining intensity or the
corresponding bands.
Asterisk
indicates double-stranded oligonucleotide;
dou-
ble asterisk
indicates single-stranded oligonucleotides;
L
50-bp DNA ladder
(Invitrogen),
C
negative control (only sense oligonucleotide),
1
and
2
result of two
representative oligonucleotide hybridization experiments (
see
Note 23
)
