Protocol for tiling amplicon generation for MinION sequencing

Version 0.1, 22nd May 2016 - initial scheme, caution likely will not amplify entire genome.

Description

This protocol for tiling RT-PCR with random hexamer priming for reverse transcription and multiplex gene specific primers for PCR. You will use two-step reagents (Protoscript II for RT and OneTaq for PCR) instead of the one-step RT-PCR you have been using until now.

Reverse transcription

  • Mix the following in a PCR tube

      2 ul Template sample 58
  2 ul Random hexamers (50 ng/µl)
  4 ul Nuclease-free H2O

  • Mix by inversion

  • Heat in the thermocycler at 65°C for 5 minutes then place on ice

  • Add the following

      10 ul ProtoScript II Reaction Mix (2X)
  2 ul ProtoScript II Enzyme Mix (10X)

  • Place on the thermocycler and start the following program

      25°C for 5 minutes
  48°C for 15 minutes
  80°C for 5 minutes

PCR

  • Make a master mix of the following:

    10 ul x 4.5 = 45 ul 5X OneTaq Standard Reaction Buffer 1 ul x 4.5 = 4.5 ul 10 mM dNTPs 0.25 ul x 4.5 = 1.13 ul OneTaq DNA Polymerase 2 ul x 4.5 = 9 ul cDNA reaction mixture 31.75 ul x 4.5 = 142.9 ul H2O

  • Mix by inversion

  • Pipette 45 ul into PCR tubes 1-4

  • Add the primers

      2.5 ul F control primer (10uM) or primer pool
  2.5 ul R control primer (10uM) or primer pool

  • PCR program:

      Step 1
  94°C 30 seconds 
  Step 2 (35 cycles)
  95°C 15 seconds
  59°C 5 minutes
 		68°C 30 seconds
  Step 3
  68°C 5 minutes

Primers

Primers starting with the name ZKV_ are Charyl’s primers and need diluting from 100 uM to 10 uM before using.

For multiplex reactions mix 1 ul of all forward and reverse primers, use 5 ul for a 50 ul reaction.

Control reactions

1.	400_7F and 400_7R
2.	400_14F and 400_14R

Multiplex reaction

Pool 1

ZKV_01F	200_4_R_2
200_6_F_1	200_10_R_2
200_16_F_1	200_20_R_2
200_26_F_3	200_28_R_2
200_33_F_1	200_36_R_2
ZKV_07F	200_43_R_2
200_47_F_3	200_51_R_2
200_55_F_1	200_59_R_2
200_65_F_1	200_70_R_2
400_21F_0	200_80_R_2
200_85_F_1	200_89_R_2
200_93_F_1	200_97_R_4
200_102_F_1	200_106_R_2
200_111_F_1	200_114_R_2
200_123_F_3	200_125_R_2
200_131_F_1	200_136_R_2

Pool 2

200_2_F_1	200_5_R_2
200_11_F_1	200_15_R_2
200_21_F_1	200_25_R_2
400_8F_0	200_32_R_2
200_37_F_1	200_40_R_2
200_44_F_1	200_47_R_2
200_52_F_3	200_54_R_2
200_60_F_1	200_64_R_2
200_71_F_1	200_76_R_2
200_81_F_1	200_84_R_2
200_90_F_1	200_92_R_2
200_98_F_1	200_101_R_2
200_106_F_3	200_110_R_2
200_117_F_1	200_122_R_2
200_126_F_1	200_130_R_2
200_136_F_1	ZKV_14R_0