Report

Aslim Taslim Benediktus Yohan Makassar 16-17 Dec 2009

Onestep Master Mix

Multiplex Master Mix for 18 sample 5x buffer 5ul x 19 = 95 ul Enzym Mix 1 x 19 = 19 ul dNTP 1 x 19 = 19 ul D1 0,25x 19 = 4,74 ul Ts1 0,125 x 19 = 2,375 ul Ts2 0,125 x 19 = 2,375 ul Ts3 0,125 x 19 = 2,375 ul Ts4 0,125 x 19 = 2,375 ul H20 14,125 x 19 = 268,375 ul

Den 1 Master Mix for 18 sample 5x buffer 5ul x 12 = 60 ul Enzym Mix 1 x 12 = 12 ul dNTP 1 x 12 = 12 ul D1 0,25x 12 = 3 ul Ts1 0,125 x 12 = 1,5 ul H20 15,625 x 19 = 187,5 ul

Den 2 Master Mix for 18 sample 5x buffer 5ul x 9 = 45 ul Enzym Mix 1 x 9 = 9 ul dNTP 1x9 = 9 ul D1 0,25x 9 = 2,25 ul Ts2 0,125 x 12 = 1,125 ul H20 15,625 x 19 = 140,625 ul

Den 3 Master Mix for 18 sample 5x buffer 5ul x 10 = 10 ul Enzym Mix 1 x 10 = 10 ul dNTP 1 x 10 = 10 ul D1 0,25x 10 = 2,5 ul Ts2 0,125 x 10 = 1,25 ul H20 15,625 x 10 = 156,25 ul

Den 4 Master Mix for 18 sample 5x buffer 5ul x 3 = 15 ul Enzym Mix 1 x 3 = 3 ul dNTP 1x3 = 3 ul D1 0,25x 3 = 0,75 ul Ts2 0,125 x 3 = 0,375 ul H20 15,625 x 3 = 46,875 ul

Run in Thermocycle
Run In PCR Pre PCR : 50 C for 30min 95 C for 15min 45 Cycles : 95 C for 30sec 55 C for 1 min 72 C for 1 min End 72 C for 10min 4 C for ----

Run In electrophoresis
Agarose 2 % - add loading dye 5 ul to cDNA 25ul - Run in 120 volt 60min

Two Step Lanciotti (using invitrogen SS III)

STEP 1 : Reverse Transcriptase o) Master Mix 1 ( 9 ul per reaction) : - DTT 1 x 18 = 18 - dNTP 1 x 18 = 18 - D2primer 0,5 x 18 = 9 - H20 6,5 x 18 = 117 Incubate 65 C 5 min

o) Master Mix 2 (5 ul per reaction): 5x first strand buffer 4 x 18 = 18 ul RNAse Out 0,5 x 18 = 9 ul Superscript III 0,5 x 18 = 9 ul Run in 25 C for 5 min 42 C for 60 min 70 C for 15 min 4 C hold

STEP 2 : Typing Master mix 22 reaction (23 ul per reaction) 10x buffer 2,5 x 22 = 55 ul dNTP mix 0,5 x 22 = 11 ul D1 0,5 x 22 = 11 ul Ts1 0,2 x 22 = 4,4 ul Ts2 0,2 x 22 = 4,4 ul Ts3 0,2 x 22 = 4,4 ul Ts4 0,2 x 22 = 4,4 ul Roche Taq polimerase 0,15 x 22 = 3,3 ul H2O 18,55 x 22 = 408,1 ul Run In 1 Cycles : 95 C for 2 min 45 Cycles : 95 C for 30 sec 55 C for 1 min 72 C for 1 min End cycles : 72 C for 2 min 4 C hold

Onestep multiplex typing Result (using qiagen)

Onestep typing TS1 TS2 Result (using qiagen)

Onestep typing TS3 TS4 Result (using qiagen)

Two step typing result

Two step typing result

Confirm onestep result

Two step

sample ID 43 57

Mksr 2,3,4 1

Jkt (-) 3

Multiplex 3&4 1&3

Ts1 x 1

Ts2 x x

Ts3 3 3

Ts4 4 x (-) 3

Ts1-Ts4

71 80 297

1,2,3 1,3 2 or 3

2 1 2

2 (-) 2

1 (-) x

2 x 2?

3 (-) 3?

x x x

not done (rna empty) (-) 2

412
502 706 2065 2145 2216 3000

(-)
1 (2) (-) 1 (3?) 1 (2?) 1 1(3)

2
2 4 3 2 2 3

2
2 4 3 2 1 3

x
1? x 1 1 x x

2?
2? x x 2? x x

x
x x 3 x x 3

x
x 4 x x 2 x

2
2 4 3 2 2 3

Two step D2 diganti dengan TS1-4

Comment
Mengenai one step RT PCR - proses kerja lebih cepat (3,5 jam) - lebih simple (karena dalam 1 tube reaksi) - sering ditemukan adanya double band yang sehingga menyulitkan analisa (mengambil kesimpulan) Two step - Proses kerja lebih lama - langkah lebih panjang - kemungkinan adanya background D1/D2 (511bp) - Band yang terbentuk lebih spesifik (jarang ditemukan double band) Beberapa sample (sample ID 43, 057, 71, 2216) masih memerlukan konfirmasi lanjutan (dengan PCR/sequencing)