Team:UCLA/Notebook/Honeybee Silk/14 July 2015

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Miniprep

Samples 1 and 3 (inoculated) were miniprepped using the Zymo Plasmid Miniprep Kit.

OD

1. 128.23 ng/uL
2. 106.85 ng/uL

Both samples were sent in for sequencing.

PCR of Lac promoter/Silk/Spycatcher

• Using primer 3 and 4 that contain overhangs with the Biobrick prefix and suffix.

Component	Volume (out of 50uL)
5X Q5 Reaction Buffer	10uL
10mM dNTPS	1uL
10mM primer 3	2.5uL
10mM primer 4	2.5uL
Template (Honeybee G-block)	1uL
Q5 High Fidelity DNA Polymerase	0.5uL
Nuclease Free Water	32.5uL

Program

Step	Temperature	Time
Initial Denaturation	98C	30s
Cycles (x25)	98C	10s
Annealing	72C	20s
Extension	72C	30s
Final Extension	72C	2min
Hold	12C	Hold

Gel Visualization and Purification

I ran the PCR product on a 1% agarose gel, expected size ~1600bp

Results: There was a band at the appropriate length and another band, probably the result of non-specific binding. The correct band was extracted and purified using the Qiagen Gel Purification kit.

OD: 88.12ng/uL

Gel Image

Double Digestion of Purified PCR Product

• The insert will be digested with EcoRI and Pst1 for preparation for ligation into the psb1c3 backbone (already digested with PstI and EcoRI).

Component	Volume (out of 50uL)
NEB Buffer 2.1	5uL
EcoRI	1uL
PstI	1uL
Purified Insert	9uL
Nuclease Free Water	34uL

Program

Step	Temperature	Time
Incubation	37C	1 hour
Heat Inactivation	80C	15 min
Hold	10C	Hold

• Ran the digestion on a gel (all 50 ul) and compared to NEB 1 kb ladder. There was a nice clean band at the appropriate size.

Gel Image

Ligation of SIlk + SpyCatcher into PSB1C3

• 3:1 insert to vector ratio.

Component	Volume (out of 50uL)
T4 Ligase Reaction Buffer	2 ul
Backbone (psb1c3)	1uL (50 ng)
Insert (silk + spyC)	10ul (120 ng)
H20	6 ul
Ligase	1uL

• Program = 16C overnight and heat inactivate 10 min

Calculating Competency

• Calculating competency of cells prepared on 7/12.
• Equation = # colonies / ug DNA plated
• Using 2 ul of 50pg/ul puc19 vector to transform in to 50 ul
• Counted 300 colonies
• plated 1x10^-5 ug (1/10 of transformation reaction).
• Therefore tranformation efficiency = 3 x 10^7 cfu/ug DNA