ITS amplification of Yeast culture
Chelex Extraction from Yeast Culture
1) Gently swirl growing culture. Pipette 50.0ul into tube.
2) Spin down at 14,000 rpm. Remove supernatant.
3) Add 200.0ul of Millipore water and pipette mix.
4) Spin down at 14,000 rpm. Remove supernatant.
5) Add 100.0ul Chelex.
6) Boil at 100.0C for 10min. 27C for 1 min.
7) Use 1.0ul for ITS amplification.
| EX Takara RXN Mixture
|
conc
|
1X
|
3
|
| Milli Q water |
|
6.15 |
18.45
|
| Takara EX Buffer |
10X |
1 |
3
|
| dNTP |
2.5uM |
0.8 |
2.4
|
| Primer Mix |
5uM |
1 |
|
| EX Taq |
5U |
0.05 |
0.15
|
| Template |
Chelex Extraction |
1 |
|
|
|
|
24
|
|
|
|
8.0ul / RXN add 1.0ul Primer and 1.0ul Chelex Extraction
|
| Well
|
Culture
|
| 1 |
S. pombe
|
| 2 |
S. cerevisiae
|
| 3 |
negative control
|
Load 2.0ul sample
Library preparation for Alien genes
Library confirmation by PCR
| Reaction mixture
|
conc.
|
1x
|
x3
|
'
|
lane
|
Primer set
|
| Template |
|
0.25 |
0.75 |
|
1 |
Primer Atitin + Btitin
|
| TopTaq Buffer |
10x |
1 |
3 |
|
2 |
Primer Atitin
|
| dNTP mix |
10 mM each |
0.2 |
0.6 |
|
3 |
AV10027
|
| Primer mix (AV10027) |
5 uM each |
0.5 |
|
|
|
|
| TopTaq |
5 U/ul |
0.05 |
0.15 |
|
|
|
| Milli Q |
|
8 |
24 |
|
|
|
|
|
10 ul |
|
|
|
|
|
|
|
|
|
|
|
| PCR cycle |
|
|
|
|
|
|
| 94C |
3 min |
|
|
|
|
|
|
|
|
|
|
|
|
| 98C |
10 sec |
|
|
|
|
|
| 60C |
3 min |
|
|
|
|
|
| x35 |
|
|
|
|
|
|
|
|
|
|
|
|
|
| 72C |
5 min |
|
|
|
|
|
load 5.0 ul of sample
| Reaction mixture
|
conc.
|
1x
|
x2
|
'
|
lane
|
Primer set
|
| Template (Adapter ligated AGM fragments) |
2.5 ng/ul |
0.5 |
1 |
|
1 |
Primer Atitin + Btitin
|
| TopTaq Buffer |
10x |
1 |
2 |
|
2 |
AV10027
|
| dNTP mix |
10 mM each |
0.2 |
0.4 |
|
|
|
| Primer mix |
5 uM each |
0.5 |
|
|
|
|
| TopTaq |
5 U/ul |
0.05 |
0.1 |
|
|
|
| Milli Q |
|
7.75 |
15.5 |
|
|
|
|
|
10 ul |
|
|
|
|
|
|
|
|
|
|
|
| PCR cycle |
|
|
|
|
|
|
| 94C |
3 min |
|
|
|
|
|
|
|
|
|
|
|
|
| 98C |
10 sec |
|
|
|
|
|
| 60C |
3 min |
|
|
|
|
|
| x35 |
|
|
|
|
|
|
|
|
|
|
|
|
|
| 72C |
5 min |
|
|
|
|
|
load 5.0 ul of sample
Control experiment (End Repair)
| Reaction mixture
|
conc.
|
1x
|
| 10kb mix (gene rich region, fragmented by fragmentase, 500 - 1,000bp) |
15 ng/ul |
18.75
|
| T4 DNA lligase buffer with 10 mM ATP |
10x |
2.5
|
| dNTP mix |
10 mM each |
1
|
| T4 DNA polymerase |
3 U/ul |
1.25
|
| Klenow DNA polymerase |
5 U/ul |
0.25
|
| T4 PNK |
10 U/ul |
1.25
|
| total |
|
25
|
|
|
|
|
20 C |
30 min
|
|
|
|
| prurified by MinElute kit |
|
|
| 12 ul of buffer EB |
|
|
| 16 ng /ul |
|
|
Control experiment (ligation)
| Reaction mixture
|
conc.
|
1x
|
| 10 kb mix (gene rich region, end repaired) |
16 ng/ul |
3
|
| Atitin + adapter |
50 nM |
0.2
|
| Btitin + adapter |
50 nM |
0.2
|
| T4 DNA lligase buffer with 10 mM ATP |
10x |
1
|
| T4 DNA ligase |
400 U/ul |
1
|
| MilliQ |
|
4.6
|
| total |
|
10
|
|
|
|
4 C |
o/n
|
|
Alien genes
