CHTSB protocol id: cxcl1
Description: Phizicky lab's pcr and ligase independent cloning for yeast protein complexes
PCR
CM (common mix)
ddH2O
1X Pfu ultra HF rxn buffer
100 ng/50ul BY4700A Template DNA
0.25 mM dNTPs (Roche Cat#1-969-064)
2.5 U Pfu Ultra Polymerase (Stratagene Cat# 600384)
Total
0.7 uM forward primer
0.7 uM reverse primer
1
2
3
4
5
6
7
50 uM
50 uM
Stock
--10X
50 ng/ul
25 mM
2.5 U/ul
ul/rxn
40.20
5.00
2.00
0.40
1.00
48.60
0.70
0.70
50.00
Thaw everything on ice and do a quick,low speed spin 1 min @ 1500 rpm
Add ingredients in order of the list above in a CM tube
Pour CM into a solution basin for mc pipetter
aliquot 48.60 ul per sample in each well of PCR plate
add primers (add .7ul of each forward and reverse) from primer plate
Seal the plate with PCR mat (LPS Cat# T351000 www.LPSinc.com)
run program :
Step 1
2
3
4
95C 2 min
95C 30 sec -> 54C 45sec -> 72C 6:30 min
95C 30 sec -> 62C 45sec -> 72C 6:30 min
72C 10 min--> hold at 4C hold
8 Gel purify pcr products and run analytical gel
***************************************************
Cut Vectors: want between 0.03-0.1 pmol of vector
Vector Name
T4 Buffer
dGTP
DTT
Vector
ddH20
T4 DNA Polymerase
Total
Stock
10X
25mM
100mM
5.0
9.6
0.4
20.3
ul/rxn
2.0
2.0
1.0
1x
5x
29x
1x
Add vectors to tubes
Make Common Mix (CM), adding T4 DNA polyermase last
Aliquot CM to each vector tube
Tap mix and then spin down briefly
Incubate at 22C for 40 min
Incubate at 75C for 40 min
Check dNTP!
***************************************************
T4 Polymerase treatment
Samples:
Stored where:
Total # Inserts:
Want between 0.12-0.48 pmol of insert
Common Mix:
T4 Buffer
dCTP
DTT
T4 DNA Polymerase
(VWR cat# 80030690)
Stock
10X
25mM
100mM
Total
ul/rxn
2.0
2.0
1.0
0.8
5.8
*Note: 2ul of insert reaction are needed per vector to anneal
EXAMPLE 96-WELL PLATE:
A:
B:
C:
D:
E:
F:
G:
H:
1
5/9.6
14.6
5/9.6
2
5/9.6
5/9.6
5/9.6
First number:
Second number:
3
5/9.6
5/9.6
5/9.6
4
5/9.6
14.6
5/9.6
5
6
5/9.6
7
5/9.6
8
9
2/12.6 14.6
5/9.6
5/9.6
5/9.6
5/9.6
10
11
12
10/4.6 2/12.6 5/9.6
5/9.6
10/4.6
Add indicated amount of DNA
Add indicated amount of ddH20 (DNA+ddH20 should add up to 14.6 ul)
check dNTP!!
1 Make common mix without polymerase
2 If needed, add water to wells
3 Add needed amount of gel purified DNA to each well
4 Add polymerase to CM
5 Aliquot CM to eppie/plate and pipet mix
6 If in plate, seal with aluminum and put extra foil around edges to prevent evaporation
7 Incubate @ 22C for 30 mins (sit on bench)
8 Heat to inactivate @ 75C for 30 mins
***************************************************
Annealing:
**All prep done @ room temp**
1 Spin down samples
2 Mix 1ul T4 treated vector and 2 ul T4 treated insert
**make sure to include a cells only sample and controls
3 Incubate @ 22C for 5 min
4 Add 1ul of 25mM EDTA
5 Incubate @ 22C for 5 min
6 Move onto transformation or store at 4C
***************************************************
Transformation to Novablue Cells
Samples:
1
2
3
4
5
6
7
8
Put the annealed samples on ice
Add 20ul of Novablue competent Cells to the annealed samples (on ice)
Tap mix and incubate on ice for 5 min
Heat 35 sec @ 43C
Ice again for 2 min and then remove from ice
Add 125ul SOC media
Plate 100ul on LB+amp plate
Incubate @ 37C O/N