PCR Method
Overall
- Day 1: DNA extraction
- Day 2: Run and optimize PCR
- Ingredients:
- IDT primers (try both sets)
- PCR Reagents
- Ingredients:
- Day 3: Run gel and image
- Ingredients:
- 1 kb ladder
- Dye
- gel dye
- PCR product
- Ingredients:
Tissue processing- keep it frozen according to Danyon "DNA tends to be stable enough and there aren’t a lot of proteins trying to degrade it, so it can be thawed without majorly compromising its integrity. However, keeping things frozen will prevent proteins from interacting with the DNA and potentially breaking it down. This issue is more important for RNA, but the principle still holds true for DNA. Since the protocol you have works with frozen or fresh tissues, it tends to be good practice to keep it frozen because the tissue is easier to break (if you were going to grind it) and the DNA won’t degrade as easily."
7/18/2024: DNA extraction of caddisfly silk samples to begin PCR
People present: Sophia
Materials:
- Caddisfly samples (silk gland AF-23-SG)
- 2-Mercaptoethanol
- Zymo kit
- Vortexer
- Centrifuge
Protocol → Deviation from protocol: no bashing beads machine instead used vortex for 1 minute
Results:
FAILURE :(- tubes labeled with 23SG-SX-7/18/24 For 119.2 ng/uL there is some kind of DNA/RNA at 260/280 so there is something there. It might be usable for PCR. We need to remeasure the second one due to negative concentration.
Possible problems:
-
Use of vortexer for 30 minutes instead of the prescribed bead basher does not fully shred the silk glands open (see below image)
- Solution: asked Josh to use his lab's bead basher, use that (scheduled for 3PM on 7/19/24)
-
Nanodrop shows there are clearly contaminants
- Solution: multiple centrifugations to remove buffer
Tasks for next time:
- use beadbasher instead of vortexer
- contaminants need to be removed more often (second 3 minute centrifugation until no liquid collects into collection tube for the final step before elution)
- remeasure the negative concentration sample and get an actual measurement
7/19/24: Attempt 2 of DNA extraction of caddisfly silk samples to begin PCR
People present: Sophia
Materials:
- Caddisfly samples (silk gland AF-24-SG)
- 2-Mercaptoethanol
- Zymo kit
- Bead basher (courtesy of josh)
- Centrifuge
no deviations from the protocol
Results:
: remeasured negative concentration from 7/18/24
7/28/24: PCR Attempt
PCR Attempt: Gel
People present: Alex
Results:
Conclusion: Fail :(
Notes: Timp + Lucy suggested we continue with this have 2 pots on the stove, wet lab ultimately decided against due to lack of resources and funding.
Synthesis Method
9/28/24: Construction of pBS+motif1 Strain
People present: Siyona, Sophia, Katie (who is this DIVA 💜)
Goal(s):
- Restriction digest motif with BamHI and XhoI
- T4 Ligase + motif + incubation 22C
- Transformation into DH10B cells -> plate on carb and incubate for 24 hours
Reconstituted motif1
- 4ug DNA -> +20 uL for 200 ng/uL concentration
- 5 uL of 200 ng/uL DNA (total 1 ug of DNA)
- 1 uL BamHI
- 1 uL XhoI
- 5 uL rCutsmart Buffer
- 38 uL H2O
labeled digest motif 1
- Run for 1 hour at 37C then 20 minutes 65C
Ligation
- 2 uL T4 ligase buffer
- 10 uL motif1
- 1 ug pBS
- 5 uL
- 1 uL T4 ligase
- (to 20 uL H2O)
labeled ligase motif 1
- Room temp for 1 hour + 10 min 65C inactivation
Transformation
- DH10B cells
- Plated on LB+Carb plates
9/29/24: Liquid Culture Setup
People present: Sophia, Kalen
Liquid Culture set up at 6PM
- Used P1000 to pick 3 colonies and incoluate LB+Carb liquid media
9/30/24: Miniprep of pBS+motif1
Miniprep of liquid culture
People present: Siyona, Sophia
Started at 10AM (14 hour mark)
Following protocol: https://www.neb.com/en-us/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010
Nano Dropped results below:
- Colony 1: 141.8 ng/uL, 1.93 A260/280
- Colony 2: 266.6 ng/uL, 1.89 A260/280
- Colony 3: 553.9 ng/uL, 1.89 A260/280
- Curves look good noticeable peak at 260 wavelength
Submitted to plasmidsaurus order HXFNPR