Fragmentation, End Repair, & A-Tailing

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• LPB (Library Prep Buffer)
• LPE (Library Prep Enzymes)
1. Prepare the following consumables:
• LPB—Vortex to mix, and then centrifuge briefly. Keep on ice.
• LPE—Pipette to mix, and then centrifuge briefly. Keep on ice.
2. Save the following cDNA Fragmentation program on the thermal cycler:
• Set the lid temperature to 105°C
• Hold at 4°C
• 30°C for 6 minutes
• 65°C for 30 minutes
• Hold at 4°C
3. Run the cDNA Fragmentation program to prechill the thermal cycler block to 4°C and preheat the lid to 105°C.
1. Combine the following volumes to prepare Library Prep Master Mix.

Reagent

Volume Per Reaction (μl)

4.4X Reaction Master Mix (µl)

8.8X Reaction Master Mix (µl)

LPB

4

17.6

35.2

LPE

6

26.4

52.8

Total

10

44

88
2. Vortex to mix, and then centrifuge briefly.
3. Add 10 μl Library Prep Master Mix to each tube containing 40 μl of cDNA.

The total volume is 50 μl.

4. Vortex to mix or pipette mix 10 times at the 25 µl stroke.

Make sure to mix completely.

5. Centrifuge briefly. Place the samples on ice.
6. Place on the prechilled thermal cycler, and then use the skip step function to run the next steps of the cDNA Fragmentation program.
7. When the thermal cycler program reaches 4°C, immediately remove the sample and proceed to the next step.