Ligation Calculations • Restriction Sequence-Ligation-Dependent Cloning • Optimized cloning efficiency is 50 ng of vecto

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Ligation Calculations
• Restriction Sequence-Ligation-Dependent Cloning
• Optimized cloning efficiency is 50 ng of vector with 2-fold excess of insert
• Recommended Insert:Vector Molar Ratio = 2:1
Insert mass(ng)= (50ng X 4000bp/ 2000bp) X 2/1
= 200ng
Vector length pGEM-4Z = __2000_____ bp
Insert length Luciferase [LUC] Insert = ___4000____ bp
LUC-INSERT
Vector mass (ng)
50ng
Vector length (bp)
2000bp
Insert length (bp)
4000bp
I: V ratio
2:1
Insert Mass (ng)
200ng
Calculate the volumes (μL) to set up a 10 μL Ligation Reaction in the table below:
Calculate the concentration of the fragments in lanes 2, 5, & 6 2 W N 1 1Kb DNA Ladder 4Z-E1 4Z-E4 3 456 Mass (ng) --- 43 33 33 40 26 100 Luc-E1 Luc-E2 37 27 33 33 Kilobases 48,- 39= 10 4 Kb Ref. Band 2 kb 8- 6- 5 4 3 2- 1.5- 1- 0.5- 1 2 3 4 5 6 7 Quick Load 1 Kb Extended NEB 50 ng/μL Mass (ng) Kilobases 45 33 33 40 33 26 100 37 27 33 33 BOB O86B43 ||||||| 2- 1.5- 1- 0.5-