Instructions:
Use nanodrop to measure concentration of pEntry vectors, make 5 femtomolar working solution of each pEntry.
Combine into 1 aliquot:
- 1uL of 5 fmol of pENTR_L4_Promoter_R1
- 1uL of 5 fmol pENTR_L1_Gene_L2
- 1uL of 10 fmol pDEST_R4_R2
- 3uL Total
WARNING: KEEP ALIQUOTED LR CLONASE MIX AT -80 AT ALL TIMES!
Add 0.5uL LR Clonase Enzyme
Additionally, remember to mix your reactions well after all elements have been added (use a 10 uL pipette set to 3 or 4 uL, then just pipette up and down gently)
Leave at room temperature for minimum of 16 hours, maximum of 24 hours.
transform just 1 uL of the reaction (This should result in around 50-500 colonies (more often than not closer to 500) with high (~90-95%) efficiency)
or storage -20C freezer.