Pro Tips
- Run at the larger scale
- ALWAYS kill it with PRoteinase K
- Transform 4 uL of the reaction
- Use ALL the transformation tricks
- Plate ALL of it
- ALWAYS run a PUC19 control for transformations
Instructions for newbies:
1.5 uL of 10 fmol/uL Dest
1.5 uL of 5 fmol/uL promoter
1.5 uL of 5 fmol/uL gene
1.5 uL of H2O
1.5 uL of LR clonase
Pipette up and down. Incubate at room temperature overnight.
Next day: Add 1.5 uL of proteinase K. Incubate for 15 minutes at 37C, then transform.
Instructions:
USE 3x VOLUME OF EVERYTHING (at least for now)
Use nanodrop to measure concentration of pEntry vectors, make 5 femtomolar working solution of each pEntry.
Excel sheet set up to calculate the necessary volumes
LR (concentration) calculations.xlsx
Sample calculation:
| DNA | DNA length from Geneious (kb) | Conc (ng/ul) | Final Vol of working solution (ul) | Conc of working solution (fM) | Vol of DNA to add, V (ul) | Vol EB (ul) | |
| l | x | y | f | 0.66*y*l*f/x | |||
| pENTR_LilrB2 | 4.4 | 200 | 100 | 5 | 7.26 | 92.74 | |
| pENTR_PirB | 5.1 | 200 | 100 | 5 | 8.415 | 91.585 | |
| pENTR_Hef1a | 3.9 | 200 | 100 | 5 | 6.435 | 93.565 | |
| pDEST | 7.9 | 200 | 100 | 10 | 26.07 | 73.93 |
Combine into 1 aliquot:
- 1uL of 5 fmol/uL of pENTR_L4_Promoter_R1
- 1uL of 5 fmol/uL pENTR_L1_Gene_L2
- 1uL of 10 fmol/uL 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.