Vector preparation:
Digest 4-6µg of vector with
appropriate enzymes (see restriction digest protocol)
If double digest not
possible purify reaction with PCR-Purification Kit after first digest, then do
second digest.
Example: 12µl plasmid =
5µg
4µl
10xNEBuffer 2
4µl
10xBSA
1µl
NheI 10U/µl
0.5µl
XhoI 20U/µl
18.5µl
H2O
Incubate 1.5 – 2h at
37¡C. For
cloning excessive reaction time can lead to exonuclease
activity.
CIAP treatment after digest:
can be done in NEB restriction enzyme buffer without adding new buffer.
add 2µl of CIAP 1U/µl: (stock CIAP 20U/µl, dilute with
CIAP dilution buffer)
for sticky ends incubate 10min at 37¡C
for blunt ends incubate 5min at 50¡C
If restriction digest cuts
out fragment smaller than 100bp vector can be purified with either
PCR-Purification Kit or with Gel Wizard Purification Kit (useful if elution
needs to be done in less than 50µl)
If fragment is bigger,
separate on gel, cut out appropriate band and purify with Promega
Gel Wizard
Insert preparation:
If PCR amplified (see
protocol) and only one single band visible on gel, purify with PCR-Purification
Kit
If PCR not very clean and
produced several bands separate on gel, cut out appropriate band and use Gel
Wizard
Digest insert with
appropriate enzymes (see above)
PCR product: clean and
concentrate with gel wizard columns, elution volume 15-50µl (no gel
purification)
If insert cut out from
another plasmid separate on gel, cut out band and gel purify with Gel Wizard
Check 2µl of purified vector
and insert for concentration by running on gel
Estimate amounts and make
sure to have one single band each, at appropriate size.
Estimation of amount on gel
with kb-ladder: load 0.5µg of DNA ladder, 3kb band=125ng
Ladder should be prepared as
follows:
Kb ladder: stock 0.5µg/µl
(-20¡C): 60µl
stock
50µl
6XDNA sample buffer
190µl
H2O
final concentration 0.1µg/µl, store at 4¡C
load 5µl = 0.5µg, if concentration of ladder different adjust
volume accordingly
Ligation:
Use 3:1 molar ratio of
insert to vector taking into account the molecular mass of both
(size
vector / size insert)/3 = factor F
amount of vector / F = amount of insert
Rapid DNA Ligation Kit (read manual before using)
For a 20µl reaction donÕt
exceed 200ng of total DNA
Vector x
ng
Insert x
ng / F
DNA dilution Buffer 2µl
H2O to
10µl, mix
T4-Ligase Buffer 10µl, mix
T4-Ligase 1µl
Control:
vector without insert to check for re-ligation rate of vector
Incubate 5-15min at room
temperature
If vector/insert/DNA dilution Buffer mix exceeds 10µl increase volume to 20µl
adjusting the volumes accordingly (4µl DNA Dilution Buffer+vector+insert
+H2O to 20µl), add 20µl T4-Ligase Buffer +1µl T4-Ligase and incubate
30min at room temperature (see manual)
Use 2-5µl for transformation
of 50µl competent cells (DH5 alpha subcloning grade)
Anne Schohl, 2008