Team:iTesla-SoundBio/DigestingPsb1c3forBiobrickingPurificationGelandGelExtractionProtocolTemplate

Red text are things to keep in mind that you may or may not need to include in the actual lab notebook.

Highlighted text are things that you need to fill in or change for your particular lab notebook depending on the variables, and concentrations.

 

Digesting Biobricking Backbone (psb1c3):

Note: All steps in this process are very rough on the DNA. This includes being surrounded by enzymes, being in LD, being in a gel, and being in buffers. Therefore, all steps in this process should be completed as quickly and efficiently. Don’t rush, but don’t go out for ice cream in between steps (did that once sorry lol).

 

Materials:

-2.1 Buffer (10X) [Frosty, “SoundBio Enzymes Box on lower shelf]

-miniprepped psb1a3    [            ]

-EcoRI and Pstl [Frosty, “SoundBio Enzymes Box on lower shelf]

-Rnase free water [Back table by microwave]

 

Procedure:

**Power on thermocycler before starting digest reaction.

1. Label the pcr tubes:
1. Starting from the tab on the left:          

2. Add the following to the pcr tube in the order in which they appear:

*Volumes of reagents determined by NEB Double Digest Finder <a style="text-decoration: none;" href="https://nebcloner.neb.com/#!/redigest">https://nebcloner.neb.com/#!/redigest</a> )

 

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Digesting psb1c3 with biobricking enzymes for future ligation with frag 1 (50 ul total)

22.9 ul diH2O 5 ul 2.1 Buffer 20.1 ul psb1c3 1 ul EcoRI 1 ul PstI

***The enzymes are very temperature sensitive, so add those quickly and at the very end!***

3. Incubate and heat in sleepy using program DIGES, in folder “SoundBio”.

For EcoRI and PstI, the program should run at room temp for 60 mins and 80 C for 20 mins.

-Started at ___

List of enzymes and inactivation temperatures for all NEB enzymes here: <a style="text-decoration: none;" href="https://www.neb.com/tools-and-resources/usage-guidelines/heat-inactivation">https://www.neb.com/tools-and-resources/usage-guidelines/heat-inactiva</a>

4. Store leftover minipreps at ______________.

 

Purification Gel of Digested Psb1c3:

Gel Purification Materials:

• TAE solution [carboy near the sink]
• 250 mL flask
• Agarose [Falcon tube on top of gel cart]
• Microwave
• Digested backbone [from previous step, should be in thermocycler]
• miniprepped psb1c3 [from the last step]
• 1 kb ladder  [Blue Citizen Salmon box located on top shelf of Sniffles]
• 5X Loading Dye [gel cart in bag]
• 20,000X Gel Stain [foil envelope in door of fridge]
• Parafilm [next to microwave]
• Scale and weigh boat
• Graduated cylinder
• Gel comb (ten wells), tray, etc.
• Power supply [on top of microwave]
• Razor
•  

Procedure:

1. Fill flask with 60 mL of TAE using graduated cylinder (120 ml for 2 purification gels, 60 ml for 1 purification gel)
2. Weigh out 0.6 g of agarose and add it to TAE
3. Cover the hole with a paper plug (paper towels)
4. Microwave TAE solution until its clear and agarose is entirely dissolved. We started with 30 seconds and did 10 second increments after.
5. Once TAE solution is sufficiently cooled, add 3 uL of 20,000X gel stain and swirl it around a bit.
6. Pour TAE solution into gel trays. Make sure they’re taped around the sides. Store any extra TAE solution in Chilly clearly labelled.
7. Once the solution has solidified, then carefully remove comb
8. Fill gel box with enough TAE to cover the gel. Make sure the gel tray rests perfectly in the middle of the gel box. Get rid of any bubbles.
9. Add the following amounts to the ten wells:
1. To get a 4:1 ratio of digest: dye, add 12.5 uL of loading dye into the digest tube. Since the wells only hold around 30 uL each, pipette 30 uL into the wells

Orientation →

Gel #1:

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1	2	3	4	5	6	7	8	9	10
3 ul ladder		30 ul Digest and LD	Add add. wells, labelled

Add additional wells if needed

 

9. Run gels (168 volts) until purple has reached halfway down the gel. Gel started running at ______ START HEATING WATER BATH TO 50 C
10. Examine gel under blue light in the bathroom. Take a picture!

 

11. Cut out bands of digested biobrick backbone. Cut off as much gel as you can from around the glowing band, but DO NOT CUT INTO BAND! *Cut at the very most two well bands in one go.*
12. Put bands in two 2 mL collection tubes for purification. Make sure to tare the tube and then weigh the gel band once it’s in there.

 

Purification Gel Expected Results: (you might do more bands and separate tubes of the same reaction, but this should be what size bands show up for the lanes holding digested p psb1a3)

[Insert Picture of gel/s]

 

Gel Extraction of BB Backbone Gel Slices:

Materials:  

• Qiagen Gel Extraction Kit [underneath bench 3 in box labelled “Qiagen Gel Extraction Kit iGEM”] Should contain:
• Buffer QG
• Buffer PE (with ethanol added)
• Buffer EB
• 2mL collection tubes
• QIAquick spin columns

• Gel slices of digested psb1c3 backbone  [From last step]
• Hot water bath [bench 4]
• Centrifuge [bench 4]
• Falcon tubes [storage closet]
• Waste container [sink]
• P1000 and P200 micropipettes [bench 3]
• Scale [bench at the back of lab]

 

Procedure:

• Taken from QIAGEN (EN)-QIAquick Spin Handbook: https://www.qiagen.com/us/resources/resourcedetail?id=3987caa6-ef28-4abd-927e-d5759d986658&lang=en</a>
• *Note: Preheat water bath to 50 C as soon as possible
• All centrifuging happens at 17,900 rcf

 

1. Add 3 volumes of Buffer QG to the gel slices. If the gel slice weighs 100 mg, for example, add 300 uL of Buffer QG.
2. Incubate at 50 C in water bath until gel slices have completely dissolved. To help dissolve gel, mix by vortexing the tube every 2-3 minutes during the incubation.
3. Place a QIAquick spin column in a provided 2 ml collection tube.
4. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min at 17,900 rcf.
5. Discard flow-through and place QIAquick column back in the same collection tube.
1. For samples larger than what will fit in the spin column, centrifuge and  repeat steps 6 and 7 until full sample has been applied and spun through column.
6. Add 0.5 ml of buffer QG to QIAquick columns and centrifuge for 1 min.
7. To wash, add 0.75 ml of buffer PE to column and centrifuge for 1 min.
8. Discard the flow-through and centrifuge the column for an additional 1 min.
9. Place column into a clean 1.5 ml microcentrifuge tube labelled appropriately with the plasmid and date.
10. To elute DNA, add 30 ul of buffer EB to the center of the QIAquick column membrane and let it sit for 5 minutes . Centrifuge for 1 min.
11. DNA should be present as a clear solution under the column.
12. Discard column.
13. Check concentration with NanoDrop.
14. Stored gel extracted digested backbone in  ____________

Results:

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Label	Concentration	Volume	Location	Notes