Nested PCR Protocol Nested PCR protokola
Nested PCR Protocol Conditions Nested PCR protokola pogoji
For 50 ul reactions, the amounts given are per reaction. Za 50 ul reakcije, so za zneske, navedene na reakcijo. Make sure you make an extra reaction for every 6 reactions to have enough master mix. Poskrbite, da vam dajo dodatno reakcija na vsakih 6 reakcije, da je poveljnik ladje dovolj premešamo.
External Round of PCR Zunanji krog PCR
Master Mix Master Mix
dNTP mix (2mM each dNTP) dNTP zmes (2mM vsak dNTP) | 5 µl 5 μl |
10x PCR buffer (15mM MgCl2) 10x pufer PCR (15mM MgCl2) | 5 µl 5 μl |
Taq polymerase (0.2U) Taq polimeraza (0.2U) | 0.2 µl 0,2 μl |
dH2O | 25.8 µl 25,8 μl |
Primer mix. Primer premešamo. Forward and Reverse primers. Naprej in nazaj oligonukleotidov. (5 pmoles/µl each) (5 pmoles / μl vsak) | 4 µl 4 μl |
Total Mastermix Volume: Skupaj Mastermix zvezek: | 40 µl 40 μl |
DNA Template DNK Predloga | 10 µl 10 μl |
Internal Round of PCR Notranji krog PCR
Nested PCR Master Mix Nested PCR Master Mix
dNTP mix (2mM each dNTP) dNTP zmes (2mM vsak dNTP) | 5 µl 5 μl |
10x PCR buffer (15mM MgCl2) 10x pufer PCR (15mM MgCl2) | 5 µl 5 μl |
Taq polymerase (0.2U) Taq polimeraza (0.2U) | 0.2 µl 0,2 μl |
dH2O | 35.8 µl 35,8 μl |
Primer mix. Primer premešamo. Forward and Reverse primers. Naprej in nazaj oligonukleotidov. (5 pmoles/µl each) (5 pmoles / μl vsak) | 4 µl 4 μl |
Total Mastermix Volume: Skupaj Mastermix zvezek: | 49 µl 49 μl |
DNA Template (Sample from external round PCR product) DNK Predloga (vzorec iz zunanjih krogu produkta PCR) | 1 µl 1 μl |
PCR Machine Cycling Parameters PCR stroj, kolesarjenje parametri | |
1 cycle: 1 cikel: | 94ºC/30 sec. 94 ° C/30 sec. |
35 cycles: 35 ciklov: | 94ºC/30 sec. 94 ° C/30 sec. |
Hold: Drži: | 4ºC 4 ° C |