Team:UCLA/Notebook/Interlab Study/14 August 2015

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PCR Amplification and Gel Extraction of InterLab Devices

PCR Reaction & Thermocycler Setup

PCR was performed to amplify the 3 Interlab gBlocks devices for increased yield to perform EcoRI/PstI double digestions for pSB1C3 cloning. Q5 high fidelity reaction mixtures were used.

Component	Amount (for 50 uL reactions)	Final Concentration
5x Q5 Reaction Buffer	10 uL	1x
2 mM dNTPs	5 uL	200 uM
10 uM InterLab Device - F Primer	2.5 uL	0.5 uM
10 uM InterLab Device - R Primer	2.5 uL	0.5 uM
InterLab Device 1/2/3 Template	0.5 uL of 1ng/uL stock	500 pg/50uL
Q5 DNA Polymerase	0.5 uL	0.02 U/uL
Nuclease Free ddH2O	30 uL	N/A

There reactions were set up (1 reaction for each Device 1/2/3 template). Templates were diluted from 10 ng/uL gBlocks stock by adding 1 uL of stock to 9 uL of MilliQ water in an eppendorf tube and stored at -20 degrees. 0.5 uL of the 1ng/uL stock was used for a final PCR concentration of 500 pg/50 uL (or 10pg/uL of reaction).

The annealing temperature calculated using the NEB Q5 Tm calculator was 72 deg Celsius, the same as the extension temperature. So, a two-step PCR was conducted instead of separate annealing/extension steps per cycle. Below is the reaction conditions setup:

Step	Temp (degrees Celsius)	Time
Initial Denaturation	98	30 seconds
30 cycles	98	10 seconds
	72	30 seconds (1kb amplicon)
Final Extension	72	2 minutes
Hold	4

After PCR was finished, the resulting products were mixed with 10 uL of 6x Loading Dye and split into two 30 uL products. Each sample was loaded into a 1% wt/vol agarose gel, along with 10 uL of 1 kbp GeneRuler DNA ladder, and ran for 20 minutes at 100V in 1x TAE buffer. (Note: only 6 lanes were on the gel, so the second 30uL product of the Device 3 PCR reaction was stored at -20, to be ran on a separate gel on Monday).

Gel was removed and imaged on an Invitrogen gel box. Pictures will be uploaded in results using cell phone camera.

Results

PCR shows high amplification of a 1kb band of each device, consistent with amplification of an expected 970 bp product in all devices using the same primer. Bands were cut and placed in eppendorf tubes. Device 1 band had a weight of 188 mg, Device 2 had a weight of 199mg, and Device 3 had a weight of 103 mg. Device 3 has a second product that has not been run on a gel yet, resulting in lower weight amounts (and possibly lower yields).

The resulting PCR shows show proper amplification. Bands were cut out and extracted using the Zymo Gel Recovery Kit, with the following protocol:

Step	Time
Cut out bands of interest from gel, and weigh the bands.
Add ADB buffer to gel pieces in eppendorf tubes in a 1:3 (wt/vol) dilution.
incubate samples at 50 degrees Celsius, shaking occasionally to ensure dissolution.	10 minutes
Transfer dissolved gels 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
Dry spin the columns at same conditions to remove excess ethanol.	30 seconds
Add 10uL 60 degree Celsius prewired Qiagen EB 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.

OD will be measured on Monday, 08/17/2015.

Directions for Monday

• Remaining Device 3 product will be ran on a gel and extracted, combined with the earlier product.
• Double digestions for each device will be conducted using EcoI/PstI.
• Resulting products will be ran on a gel and gel extracted for ligation. (Need to grab linear pSB1C3 for ligation)
• Ligation will be conducted and resulting product will be transformed in BL21(DE3) chemically competent E. coli using Zymo Mix n Go cells.
  • Cells will be plated on LB + chlor and incubated overnight.
  • Will also transform + (MeasKit) and - (pTetR) control plasmids previously miniprepped.

Directions for Tuesday

• Check devices + controls for transformants. Observed fluorescence for devices and + control to verify correct plasmid function.
• Inoculate 15 mL LB + chlor starter cultures with each device and control. Incubate shaking at 37 degrees overnight.

Directions for Wednesday

• Miniprep 3 mL of each starter culture using the Promega kit.
  • After miniprep, send each plasmid out for Genewiz sequencing using VF2 and VR primers.
  • Also, double digest each plasmid with EcoRI/PstI and run on a gel to verify size of insert is correct for all controls and devices.
• Inoculate 500 uL of 50% glycerol with 500 uL of each culture in cry tubes, and store glycerol stocks at -80 in Fasih's box.
• If all goes to plan, we will start assaying on Wednesday with the remaining cultures in a microplate reader overnight.