in-fusion+reaction+4-12-13

started planning in-fusion reaction 4-12-13 actual experiment 4-16-13

I will in-fuse the control insert into the control vector which I electroeluted and ethanol precipitated. I will also in-fuse the random pentadecamer cDNA library into the vector.

Samples cDNA 4-10-13 (cDNA constructed from random pentadecamers) (estimated conc. 248 ng/uL) Ladder 4-10-13 (56 ng/uL) Control Insert 4-10-13 (12.3 ng/uL) Control Vector 4-10-13 (10.6 ng/uL)

Control insert and vector Insert size (2 kb Control Insert): 2 kb Linearized Vector size (pUC19 Control Vector, linearized): 2686 bp

I will use In-Fusion molar ratio calculator should use 100 ng vector 148.9 ng insert I could reduce these by 4 if desired 25 ng vector 37.2 ng insert

25*"ng"*"uL"/(10.6*"ng") = 2.36 uL vector 37.2*"ng"*"uL"/(12.3*"ng") = 3.0 uL 2 uL 5X In-Fusion HD Enzyme premix 2.64 uL H20

Incubate 15 min 50 C and then ethanol precipitate

For the library reaction I will refer to the SMARTer In-Fusion directional cDNA library manual They perform 3 library reactions they all use 2 uL 150 ng/uL vector Then for the DNA: 4 uL 150 ng/uL (600 ng), 4.5 uL 150 ng/uL (675 ng), and 5 uL 150 ng/uL (750 ng)

My cDNA has a concentration of 248 ng/uL. I'll use 675*"ng"*"uL"/(248*"ng")= 2.72 uL

In-Fusion reaction 2 uL 5X In-Fusion HD Enzyme Premix 2 uL pSMART2IFD Linearized Vector (150 ng/uL) 2.72 uL of 248 ng/uL 3.28 uL H20

Negative Control Reaction 2 uL 5X In-Fusion HD Enzyme Premix 2 uL pSMART2IFD Linearized Vector 150 ng/uL 6 uL H20

Positive Control Reaction 2 uL 5X In-Fusion HD Enzyme Premix 5 uL H20 1 uL pUC19 Linearized Vector 2 uL Control Insert

Incubate at 15 min 50 C then ethanol precipitate