Team:iTesla-SoundBio/labnotebook/labnotebook060818.html

6/8/18

Juan Jose Wheeler, Juntao Zhong, Sarah Alvi, Charlie Anderson, Mary Elizabeth Adler

 

Purpose: To run a PCR on our ordered Factor C fragments with the incoming primers from IDT. (This PCR will not only make a ton of copies of our gene fragments for future procedures, but also fix the error in fragment 2 that left out the BglII cut site).Also, to grow up a colony of e.coli for the future transformation of our b.subtilis vector.

 

Materials:

-PCR tubes and tube racks [storage]

-Dessicated primers [now not dessicated, located in rectangular box in Sniffles I]

-Dessicated factor C fragments 1 and 2 gblocks  [now not dessicated, located in rectangular box in Sniffles I]

-RNA-ase free H2O [rectangular box in Sniffles I]

-Phusion Buffer [Frosty “Sound Bio Enzymes” box]

-Phusion Polymerase [Frosty, “Sound Bio Enzymes” box}

-dNTPs [Sniffles , rectangular purple box]

-ddH2O [laminar flow hood]

 

Reagents to Combine:

 

<tbody> </tbody>

Reactions:	1	2	C
Reagents:	---------------------	---------------------	---------------------
Forward Primer BB	X		X
Reverse Primer BB	X	X	X
Forward Primer 2 (corrective)		X
FC 1 (gblock)	X
FC 2 (gblock)		X	X

 

Procedure:

***Keep buffer, dNTPs, and polymerase on ice throughout procedure (or take it out of the freezer only when you’re about to use it).

1. (This step should be completed before the reagents are mixed together) On Sleepy, create a program that runs as follows (subdirectory: “standard”, program: “general”):

1. Lid temperature 100.0 C
2. 98.0 C for 10 secs
3. Loop for 30 cycles:
1. 98C for 10 secs
2. 55C for 20 secs
3. 72C for 60 secs

4. 72C for 2 mins
5. Hold at 4C
6. Label 3 PCR tubes:
7. FC 1

Ii. FC 2

Iii. Control → to tell whether our redesigned corrective primer worked

3. To convert desiccated Factor C frags into working solutions:

1.Add 20uL RNAase-free water to the vials containing Factor C fragments, thus creating 50ng/uL working solution. Vortex it.

4. To convert desiccated primer into working solution: (will need to do this for Forward Primer 2 (Fixer, but only need to do step 4.4 for biobricking primers (labelled pcbA1 forward and reverse))

1. Centrifuge primer tubes before opening, 13,000 rcf for 1 minute
2. Check label of primer tube for nmol in tube (e.g., 34.7 nmol). Multiply by 10 to get the volume of ddH2O in ul needed to suspend the primers at 100uM. (Example, 34.7 nmol of primer would be suspended in 347 ul of ddH2O). This volume will likely be unique for each primer. Be careful at this stage to not touch the inside of the tube cap and avoid setting the cap down. Just remove cap, add water, and put the cap back on.
3. Vortex well, and invert a few times to dissolve any dried DNA on the sides or top of the tube.
4. Prepare a 10uM working solution by creating a 10x dilution by adding 9 ul of ddH2O with 1ul of primer in a separate PCR tube. Label accordingly and centrifuge.

5. Pipette 35.5 ul of RNase-free water into all three originally labeled PCR tubes.
6.  Pipette 10 ul of Reaction Buffer into all tubes
7. Pipette 1 ul of 10uM forward primer into all tubes
8. Pipette 1 ul of 10uM reverse primer into all tubes
9. Pipette 1 ul of dNTPs into all tubes.
10. Pipette 0.5 ul of appropriate geneblock into all tubes
11. Pipette 1 uL of DNA Polymerase into all tubes
12. Centrifuge PCR tubes in the mini PCR tube centrifuge for five seconds.
13. Place PCR tubes in Sleepy. Record below where each tube is placed in case the label wears off: the tab of the caps is on the C side, the order of the tubes is 1, 2, C
14. Run the program created above.

 

PCR Purification Procedure: (If using Qiagen columns, use 1 wash of Buffer PE instead of these instructions. Follow these if using GeneCatch column )

 

Materials:

-Four microcentrifuge tubes [back storage]

-Collection tubes and spin columns [Qiagen kit under bench 3]

-Buffer PB [Qiagen kit under bench 3]

-Buffer PE [^^^]

-Elution Buffer [^^^]

From

<a href="https://www.qiagen.com/us/resources/resourcedetail?id=3987caa6-ef28-4abd-927e-d5759d986658&lang=en">https://www.qiagen.com/us/resources/resourcedetail?id=3987caa6-ef28-4abd-927e-d5759d986658&lang=en</a>

1. Prepare two microcentrifuge tubes labeled FC 1 (Factor C Fragment 1) and FC 2  (Factor C Fragment 2) Pipette all liquid from each PCR solution into the corresponding microcentrifuge tube.
2. Pipette 250 ul of Buffer PB into each microcentrifuge tube. Pipette up and down to mix.
3. Set up a spin column in a collection tube for each PCRed solution and label them FC 1 and FC 2. Pipette all the mixture from the microcentrifuge tubes into the spin columns.
4. Centrifuge columns at 5000 RPM for 60 seconds. Dispose of flow-through.
5. Add 750 uL of Buffer PE to wash
6. Centrifuge columns at 13,000 RPM for 60 seconds. Dispose of flow-through. Centrifuge again for another 1 min.
7. Move spin columns onto a new set of microcentrifuge tubes. Discard collection tubes.
8. Pipette 30 ul of Elution Buffer onto the center of each spin column (make sure the liquid is on the membrane). Let columns sit for four minutes.
9. Centrifuge columns at 13,000 RPM for two minutes.
10. Dispose of spin columns. Record where all materials are put back.

Inoculating LB with Colony of E.coli for tomorrow’s transformation:

Copy from Protocol book located on the top of Shaky