Team:UCLA/Notebook/Interlab Study/17 August 2015

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Digest & Ligate Assembly of InterLab Devices

OD Measurement

DNA concentration of the eluted Devices were measured using a DeNovix Nanodrop Spectrophotometer in the dsDNA app using Qiagen EB as a microvolume blank. The resulting concentrations were measured as a ng/uL unit, recording the A260/A280 and A260/A230 ratios for purity of sample. Based on calculations, the amount needed for 1 ug of each device to be pipetted was calculated for the double digests below.

Double Digestion of Devices and pSB1C3 Plasmid Using EcoRI and PstI

5 PCR tubes were prepared for parallel digestion of all three devices (3) and an aliquot of pSB1C3 plasmid for linearization (2). Double digestion reactions were prepared using the following conditions in the NEB EcoRI/PstI Double Digestion.

Component	50 uL Reaction
Template DNA (Devices, Plasmid)	1 ug
10x NEB 2.1 Buffer	5 uL (1x)
EcoRI	1 uL (10 Units)
PstI	1 uL (10 Units)
Nuclease free ddH2O	Up to 50 uL

Samples were prepared in the PCR tubes with their respective aliquots of template DNA, and placed in the thermocycler. Reactions were incubated at 37 degrees Celsius for 90 minutes, heat inactivated for 20 minutes at 80 degrees Celsius, and held at 10 degrees Celsius indefinitely.

Each sample was then mixed with 10 uL of 6x Loading Dye (1x final), split into two 30 uL samples, and loaded into 30 uL wells in a 1% wt/vol agarose gel supplemented with DNA SYBR Safe Stain (0.5g agarose, 5 uL SYBR Safe, 50 mL 1x TAE). Electrophoresis was ran at 100V for 30 minutes in 1x TAE buffer or until the loading front was 80% through the gel. Gel was removed and imaged in an Invitrogen gel box with a phone camera, and uploaded annotated below.

Ligation of digested Devices in digested pSB1C3 using T4 Ligase

Each of the three devices digested were ligated into the digested pSB1C3 vector using the NEB T4 ligase reaction in a 20uL reaction. Three PCR tubes were prepared on ice with the following reaction for the ligation.

Component	20 uL Reaction
10x T4 DNA Ligase Buffer	2uL (1x)
Vector DNA (pSB1C3)	1:3 molar ratio (pmol)
Insert DNA (Devices 1/2/3)	1:3 molar ratio (pmol)
T4 DNA Ligase	1 uL
Nuclease Free ddH2O	up to 20 uL

Samples were loaded, mixed, and placed into the thermocycler. Samples were ligated for 10 minutes at RT (22 degrees Celsius), and heat inactivated at 65 degrees Celsius for 10 minutes, and held at 4 degrees Celsius indefinitely. Samples were then removed and chilled on ice, before being transformed into chemically competent E. coli BL21(DE3) Mix n Go cells.

Transformation of Devices in pSB1C3 into BL21(DE3) Cell Lines

Cells were transformed using the Zymo Mix n Go Protocol. Three 50 uL tubes of cells were thawed on ice for 10 minutes, and each tube was added with 1uL of a respective device and mixed gently. Plates containing LB+chloramphenicol were prewarmed to 37 degrees Celsius in the incubator before plating of the transformants.

Cell/DNA mixtures are incubated on ice for 10 minutes before cells are rescued in 200 uL SOC and placed in 37 degrees Celsius shaking at 300 RPM. The entire outgrowth is then plated on the prewired plates, spread using sterile glass beads, inverted and incubated at 37 degrees Celsius overnight for 16 hours.

Plates were checked the following day for growth of colonies, check out tomorrow for observations regarding the cells.

This was performed in tandem with the transformation efficiency tests on the freshly prepared cells.

Results/Images/Observations

Initial OD of PCR samples from Friday

Sample	ng/uL	A260	260/230 Ratio	260/280 Ratio
Device 1 (10 uL)	26.24	0.525	0.35	2.01
Device 2 (10 uL)	78.70	1.574	0.23	1.99
Device 3 (10 uL)	56.10	1.122	0.98	2.00

There was not enough sample for each device to be digested in a 50 uL reaction, so the PCR was redone for each device in triplicate. The 50 uL products were then pooled into 150 uL samples, which were PCR clean-upped using the Zymo DNA Clean & Concentrator-5 Kit.

DNA Clean and Concentrator of Pooled PCR Samples

Step	Time
Add DNA binding buffer to PCR product in a 5:1 vol/vol ratio (i.e. 750 uL DNA binding buffer to 150 uL PCR sample). Vortex to mix.
Transfer bound DNA to a Zymo spin column and collection tube.
Spin columns at 20 degrees Celsius at 13k RPM to elute the buffer	30 seconds
Discard buffer in collection tube. Add 200 uL of wash buffer to each column and respin at same conditions.	30 seconds
Add a second 200uL of wash buffer to each column and respin at same conditions.	30 seconds
Add 10uL 60 degree Celsius Zymo DNA Binding buffer to each column. Incubate columns.	2 minutes
Transfer columns to eppendorf collection tubes and label tubes and caps. Spin column at same conditions.	30 seconds
Remove spin columns and cap the tubes of eluted DNA. Measure OD in nano drop for DNA concentration.

The OD of each sample are listed as below:

Sample	ng/uL	A260	260/230 Ratio	260/280 Ratio
Device 1 (11 uL)	206.26	4.125	2.33	1.83
Device 2 (11 uL)	123.01	2.460	2.02	1.93
Device 3 (11 uL)	171.91	3.438	1.92	1.91

Digestion of PCR pooled Interlab Devices

EcoRI and PStI digestions set up using the buffers and components as listed above were conducted. 1 ug of each template was digested (4.85 uL of D1, 8.129uL of D2, and 5.82 uL of D3, respectively) in a 50 uL double digest reaction using NEB Buffer 2.1, nuclease free water, the above mentioned REs, and at the above mentioned conditions in a thermocyler.

Each product was then suspended in 250 uL of DNA binding buffer (the above Zymo Clean and Concentrator Protocol) and the rest of the digest clean up was completed, measuring the OD afterwards. The OD was as follows:

Sample	ng/uL	A260	260/230 Ratio	260/280 Ratio
Device 1 (11 uL)	206.26	4.125	2.33	1.83
Device 2 (11 uL)	123.01	2.460	2.02	1.93
Device 3 (11 uL)	171.91	3.438	1.92	1.91

Ligation of Devices using T4 Ligase

Device 1:

Component	20 uL Reaction
10x T4 DNA Ligase Buffer	2uL (1x)
Vector DNA 2029 bp (pSB1C3)	1:3 molar ratio (pmol) 50 ng (0.759 uL)
Insert DNA 959 bp (Devices 1)	1:3 molar ratio (pmol) 70.90 ng (1.155uL)
T4 DNA Ligase	1 uL
Nuclease Free ddH2O	up to 20 uL (15.012uL)

Ligations were incubated at 25 degrees Celsius for 60 minutes, inactivated at 65 degrees for 10 minutes, and held at 4 degrees Celsius for stabilization. Ligations were chilled in the -20 degrees Celsius incubator. Device 2:

Component	20 uL Reaction
10x T4 DNA Ligase Buffer	2uL (1x)
Vector DNA 2029 bp (pSB1C3)	1:3 molar ratio (pmol) 50 ng (0.759 uL)
Insert DNA 959 bp (Devices 2)	1:3 molar ratio (pmol) 70.90 ng (1.94 uL)
T4 DNA Ligase	1 uL
Nuclease Free ddH2O	up to 20 uL (14.227uL)

Device 3:

Component	20 uL Reaction
10x T4 DNA Ligase Buffer	2uL (1x)
Vector DNA 2029 bp (pSB1C3)	1:3 molar ratio (pmol) 50 ng (0.759 uL)
Insert DNA 959 bp (Devices 3)	1:3 molar ratio (pmol) 70.90 ng (1.32uL)
T4 DNA Ligase	1 uL
Nuclease Free ddH2O	up to 20 uL (14.847uL)

For Rest of Week

1. Transform the three ligations into prepared BL21(DE3) chemically competent cells using the Zymo Mix n Go Protocol.

2. Pick 4 colonies from each device transformation and screen for insert size, directionality and presence using RedTaq colony PCR.

3. Overnight each colony in 10 mL LB + chlor.

4. Inoculate glycerol stocks with each of the overnight cultures and freeze.

5. Miniprep and sequence each clone using VF2/VR sequencing primers. Verify sequence during weekend.