Team:iTesla-SoundBio/labnotebook/labnotebook061118.html

6/11/18

JJ Wheeler, Charlie Anderson, Mary Elizabeth Adler, Chaitanya Koli, Ila Sharma.

 

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]

-BB Primer Reverse and Forward primer working solutions [Sniffles I rectangular box]

-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 (with the tab on the lid)	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).

2. Label 3 PCR tubes:
3. FC 1

Ii. FC 2

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

 

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 0.5 ul of appropriate geneblock into all tubes
10. Pipette 1 ul of dNTPs 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 (Subdirect: Standard, Name: GENERAL) 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. All three PCRs (FC1, FC2, and C) went in the cylindrical container with the purple lid in Sniffles. They would not all fit in the purple box.

 

Analytical Gel:

 

Materials:

-TAE Solution [carboy near sink]

-250mL flask [SoundBio storage]

-Loading Dye [gel cart by bench 1]

- 20,000X Gel stain [foil envelope in Chilly]

-Parafilm [next to microwave]

-scale and weigh boat

-Graduated cylinder [storage]

-gel comb, gel tray (10 wells) [gel cart by bench 1]

-power supply [gel cart]

-first PCR products [labelled 1, 2, and C in rectangular box in Sniffles 1]

 

Procedure:

1. Measure out 30mL TAE in a graduated cylinder.
2. Measure out 0.3 g of agarose using a weigh boat and the the scale
3. Pour 15mL from graduated cylinder into the flask.
4. Add the 0.6 g of agarose into the flask, and add rest of the TAE.
5. Microwave flask for 30 secs, and then microwave in 10 sec intervals until there is no more agarose visible, and it hasn’t started bubbling. Make sure to place some paper towel in the open end of the flask so it doesn’t bubble over.
6. Allow flask to cool, swirling with a glove every couple minutes to prevent solidification.
7. Pipette 1.5 uL of 20,000X gel stain into the cooled flask.
8. Pour contents of flask into gel tray until it reaches the tip of the gel tray to avoid flowover and wait for it to solidify (~30 minutes). Tape the ends of the gel tray to prevent the solution from flowing out. Remove tape and gel comb after solidification.
9. Place gel tray in gel box and fill gel box with TAE solution until gel tray is submerged.
10. Pipette the following reagents (in table) onto parafilm in the order they will appear on the gel. Pipette up and down to mix. Take care to remember which tube corresponds to which droplet.
11. Load from parafilm into wells using the following diagram:

<tbody> </tbody>

1	2		3	4	5	6	7	8
3uL 10kb ladder + 3uL LD	3uL pcr purified 1 + 2uL LD		3uL pcr purified 2 +2uL LD	3uL pcr purified C + 2uL LD

 

12. Connect to power supply and turn it on at 140 V
13. Wait until purple band is ⅔ of the way down the gel and then shut off
14. Once gel has finished running, view it under the blue light reader.

 

Picture of gel for 6/11/2018: