Project:
Date:_______
Name:______
1, Gel extraction
QIAEXII gel extraction kit, Qiagen, Cat#: 20021
> Add 500 ul of Buffer QX1 and 5 ul of QIAEX beads to slice of gel
(Gel size is not matter. Simply add 500 ul of the buffer without weighing)
(Add some more buffer or 3MNaOAc when the buffer turn into pink)
> Incubate at 50 C until gel piece melts completely
> Centrifuge for 5 sec at maximum speed
> Wash the pellet with 500 ul of QX1
> Wash the pellet with 500 ul of buffer PE twice
> Air-dry the pellet for 20 min until the pellet becomes white
> Add 5 ul of Tris pH8.0 buffer (EB buffer from QIAprep mini prep spin kit, Qiagen
Cat#: 27104)
> Voltex, spin down
> Take 3 ul of supernatant
2, Ligation into cloning vector
pGEM T easy vector, Promega, A1360
> Ligatin reaction
PCR product: 3 ul
Vector:
0.5 ul Lot #:________
2x buffer:
3 ul
T4 ligase:
0.5 ul
Total 7 ul
> Mix well by pipetting (important)
> Incubate at 4C for overnight
3, Transformation
DH5alpha competent cell, subcloning efficiency (Invitrogen, Cat#: 18265-017)
SOC media (Invitrogen, Cat#: 15544-034)
Mix ligation product and competent cells
Ligation product:
3 ul
Competent cells:
60 ul
> Incubate at 4 C for 30 min
> Heat-shock at 42 C for 30 sec
> Put it back on ice immediately, on ice incubation for 1 min
> Add 200 ul of SOC media
> Incubate at 37 C for 30 min
> Spread entire cells onto ampicillin plate
> Incubate plate at 37 C for 16 hrs