in-fusion reaction 4-12-13

2013-12-01

azim58 - 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

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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

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