BioBootCamp
BioBootCamp kdorfman Tue, 06/20/2017 - 15:482016 Bio Bootcamp
2016 Bio Bootcamp kdorfman Wed, 06/29/2016 - 17:19
2017 bio boot camp
2017 bio boot camp kdorfman Tue, 06/20/2017 - 18:0224 students
- pub med search
- BLAST
- pipetting
- Serial dilution
- calculations
- DNA extraction (G&GA protocol)
- Quantification by nanodrop
- gel electrophoresis - w/ & w/o RNAse
- wide range marker
2018 Bio Boot Camp
2018 Bio Boot Camp kdorfman Mon, 07/02/2018 - 22:432022 Bio Boot Camp
2022 Bio Boot Camp kdorfman Wed, 06/22/2022 - 18:20FIRST DAY
Pipetting Exercise
Weigh 20 uL, 200 uL, 1000 uL to check pipets
Enter data into pipet spreadsheet
DNA Extraction
Extract DNA from fish fin clips with HotSHOT DNA extraction prep
- 50 uL alkaline lysis reagent per rxn
- 50 uL Neutralization buffer per rxn
- Aliquot 1 mL of each per pair
Rolf brings 20 fin clips
Students work in pairs, doing 4 clips per pair.
Set up PCR reactions
Dilute primers from Rolf's lab for GFP (final 0.4 uM in mix)
- Each 25 uL reaction contains:
- 12.5 uL 2X Master mix
- enough primer to make 0.4 uM
- 5 uL DNA
- water to 25 uL
- We make 900 uL Master Mix with primers
- 3.6 uL each 100uM primer
- 450 uL 2x master mix
- 446.4 uL water (to final volume 900 uL)
- Each individual student tests all 4 of the DNA samples
- 20 uL per rxn
- aliquot 180 uL per pair
Make enough master mix for 9 reactions per pair
PCR program (finclip in Main menu):
| Step | time (min) | temp | comment |
|---|---|---|---|
| 1. Initial denaturation | 2 | 94 | |
| 2. denaturation | 0.5 | 94 | |
| 3. annealing | 0.5 | 55 | ~4C below primer Tm |
| 4. extension/elongation | 0.5 | 72 | or one min per kb 1 |
| 5. go to 2 | 38 times | ||
| 6. final extension | 5 | 72 | |
| 7. hold | forever | 15 | can cut short and put into fridge |
SECOND DAY
Gel
- 1 1% agarose gel per pair
- SYBR Safe 1 uL/10 mL agarose
- small gel (50 mL)
- ? small tooth comb, 2 per gel
- 1X TAE
- loading dye
- DNA standard
BLAST
Use primer sequences?
Analyze Gel
- check bands in blue light
- take cell phone photo
- send to Rolf with documentation
-
45 sec is better for mcherry; can use 45 sec to 1 min for GFP. EGFP and mcherry amplicons are ~500 BP ↩︎
Master mix
Master mix kdorfman Mon, 06/12/2023 - 15:052023 Boot Camp
2023 Boot Camp kdorfman Wed, 06/21/2023 - 18:53- streamlined pipet exercise
- Genotyping of Rolf's fish
Zebrafish finclip genotyping
Zebrafish finclip genotyping kdorfman Wed, 06/21/2023 - 18:56Copied from 2022:
*DNA Extraction**
Extract DNA from fish fin clips with HotSHOT DNA extraction prep
- 50 uL alkaline lysis reagent per rxn
- 50 uL Neutralization buffer per rxn
- Aliquot 1 mL of each per pair
Rolf brings 20 fin clips
Students work in pairs, doing 4 clips per pair.
Set up PCR reactions
Dilute primers from Rolf's lab for GFP (final 0.4 uM in mix)
- Each 25 uL reaction contains:
- 12.5 uL 2X Master mix
- enough primer to make 0.4 uM
- 5 uL DNA
- water to 25 uL
- Karlstrom recipe for 900 uL Master Mix with primers: (see Master mix calculator)
- 3.6 uL each 100uM primer
- 450 uL 2x master mix
- 446.4 uL water (to final volume 900 uL)
- Each individual student tests all 4 of the DNA samples
- 20 uL per rxn
- aliquot 180 uL per pair
Make enough master mix for 9 reactions per pair
PCR program (finclip in Main menu) (don't know which thermocycler)
Thermocycler 3 in 364: folder BIOBOO: programs HOT92, ZFPCR
| Step | time (min) | temp | comment |
|---|---|---|---|
| 1. Initial denaturation | 2 | 94 | |
| 2. denaturation | 0.5 | 94 | |
| 3. annealing | 0.5 | 55 | ~4C below primer Tm |
| 4. extension/elongation | 0.5 | 72 | or one min per kb 1 |
| 5. go to 2 | 38 times | ||
| 6. final extension | 5 | 72 | |
| 7. hold | forever | 15 | can cut short and put into fridge |
SECOND DAY
Gel
- 1 1% agarose gel per pair
- SYBR Safe 1 uL/10 mL agarose
- small gel (50 mL)
- ? small tooth comb, 2 per gel
- 1X TAE
- loading dye
- DNA standard
BLAST
Use primer sequences?
Analyze Gel
- check bands in blue light
- take cell phone photo
- send to Rolf with documentation
-
45 sec is better for mcherry; can use 45 sec to 1 min for GFP. EGFP and mcherry amplicons are ~500 BP ↩︎