7. PCR產物與TA載體連接
pGEM-T vector is T-tailed at the insert site. To improve the ligation
efficiency, it is recommended PCR product be A-tailed.
+A system
Purified PCR product 6.1ul
dATP(1mM) 2ul
10×Taq buffer 1ul
MgCl2 0.6ul
Taq 0.3ul
vortex, then incubate at 70C for 15~30min
Ligation with pGEM-T vector
1. Briefly centrifuge the pGEM-T and Control Insert DNA tubes to collect
contents at the bottom of the tubes.
2. Set up ligation reactions as described below. Note: Use 0.5ml tubes known
to have low DNA-binding capacity (e.g., VWR Cat.# 20170-310).
3. Vortex the 2X Rapid Ligation Buffer vigorously before each use.
Ligation System
2×rapid reaction buffer 5ul
pGEM-T vector 0.5ul
+A product 3.5ul
T4 DNA ligase 1ul
Vortex, then incubate at 25C for 1h. Ligation product can be use
directly for transformation or store at -20C
連接溫度及時間：TA載體可快速連接，2h即可，25C，也可4C過夜。表達載體連接16C 3h后即可轉化，或4C/16C連接過夜。
Transformation and selection of the target clone
a. 取出感受態菌室溫放置至半融狀態。
b. 加入5μl質粒(若為連接產物，取5ul轉化；若為純質粒，取1ul轉化)，輕攪至混勻。冰浴30分鐘。
c. 42℃熱休克45－50s，注意熱休克時不能攪拌或振動。
d. 冰上放置2－3min。
e. 加入500μl 無抗生素 LB液體培養基，37℃搖床培養30min。
f.
取菌液300μl涂平板，37℃倒置培養12－16h。(如是純質粒轉化，取50ul涂板即可；如是連接產物轉化，可10000rpm離心后，留100ul上清取50-100ul涂板)
Transformation efficiency of TA vector is relatively higher than that of
double-enzyme-digestion ligation. So 100ul LB after incubation should be
enough to spread on the plate.
100µl of 100mM IPTG and 20µl of 50mg/ml X-Gal may be spread over the surface
of anLB ampicillin plate and allowed to absorb for 30 minutes at 37°C prior to
use. Spread IPTG first on the plate, then X-Gal. There will be precipitates,
If mix the two in a tube.
For TA clone, after incubate at 37C overnight, place the plate in 4C for 2~5h
to identify the target clone.
Successful cloning of an insert in the pGEM-T Vector interrupts the coding
sequence of β-galactosidase; recombinant clones can usually be identified by
color screening on indicator plates. Those inserted clones are white, the
others blue.