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Team:Stanford/Notebook/Lab Work/Week 6
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'''Suggestions from Christina:''' | '''Suggestions from Christina:''' | ||
| - | + | *diagnostic gels from yesterday's digestions (done by Francisco) | |
| - | + | run promoters (pBAD, pLUX) on 0.8%, RSID's (1 and 2) and sRNA's (1, 2, 1C, 2C) on 2% | |
| - | + | gel extract everything (on separate gels), depending on how complete digestions look- if PCR cleanup, SIP treat | |
| - | + | *PCR cleanup inserts (PCR assembled parts) | |
| - | + | use MiniElute columns | |
| - | + | use 10 mM Tris instead of H2O (water here is acidic, and elution is pH-dependant) | |
| - | + | let sit 1 minute before eluting | |
| - | + | load 1 uL on diagnostic gel | |
| - | + | *check enzymes to see if getting low: order more if necessary | |
| - | + | *colony PCR | |
| - | + | primers: complementary to prefix and suffix or VF and VR? (VF and VR- further from ends to allow subsequent sequencing) | |
**expected lengths with and without insert: ladder choice, gel %, extension time | **expected lengths with and without insert: ladder choice, gel %, extension time | ||
| - | + | 10 uL reactions, run 5 uL on gel | |
| - | + | setting up reactions: | |
**make, aliquot master mix (SuperMix + primers) | **make, aliquot master mix (SuperMix + primers) | ||
**pick colony with innoculating needle: inoculate PCR reaction, then streak on small square or wedge on a plate (make sure plates are dry) | **pick colony with innoculating needle: inoculate PCR reaction, then streak on small square or wedge on a plate (make sure plates are dry) | ||
**screen (PCR) 5 colonies to start, but streak out 10-15 extras just in case/for further screenings | **screen (PCR) 5 colonies to start, but streak out 10-15 extras just in case/for further screenings | ||
| - | + | if no bands on gel, could be: not enough cells, not correct plasmid | |
| - | + | if positive results, get sequenced | |
== 8/5 Thursday == | == 8/5 Thursday == | ||
Revision as of 21:50, 4 August 2010
| Home | Project | Applications | Modeling | Parts | Team | Notebook |
Spring: Brainstorming | Spring Meetings
Summer: Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Summaries
|
8/2 Monday
Greg's Notebook
- Inoculated freezer stocks of most of our parts with Alex
- Began planning for promoter characterization project:
- Going to do 3A ligations of the form: promoter + superfolded GFP + Kan plasmid
- Process:
- Digest parts with at least 2 ug DNA
- PCR cleanup
- Diagnostic gel (remember to save uncut DNA for control)
- Ligate in PCR tubes
- Heat inactivate at 65 C for 20 min
- Performed first digestion (F2620 + sfGFP + pSB4k5)
| Part # | Part amount | BSA | NEB (#, amount) | Enzymes (1 uL each) | Water |
| F2620 | 10.8 | 3 | 2, 3 | E + S | 11.2 |
| pSB4k5 | 20 | 3 | 3, 3 | E + P | 2 |
| sfGFP | 11.76 | 3 | 2, 3 | X + P | 10.24 |
- Let digestions run for 1 hour at 37 C
- Ran diagnostic gel:
- sfGFP digested nicely, F2620 yielded 3 bands corresponding to insert, cut plasmid, and uncut plamid
- pSB4k5 gave only one band and a smudge, so I decided to redigest with only .5 uL of each enzyme, leaving it to run overnight
8/3 Tuesday
Greg's Notebook
- Ran diagnostic gel of overnight pSB4k5 digest
- Still didn't work; there were 3 bands, the smallest of which was about the size of the plasmid backbone, and there was no band corresponding to the ccdB insert
- Decided to re-redigest pSB4k5 using .5 uL of each enzyme for only 1 hour
- After running on a diagnostic gel, discovered that this, too, failed in the same way as the previous digestions had. I decided to try other Kan plasmids
- PCR'd Chris's transcription factor promoter (pTFc)
- Ran nanodrop of pTFc and yesterday's sfGFP and F2620:
| Part # | 230 | 280 | Concentration |
| pTFc A | 1.36 | 1.79 | 196.1 |
| pTFc B | 2.08 | 1.86 | 159.7 |
| sfGFP | 1.89 | 1.87 | 52.9 |
| F2620 | 1.82 | 1.82 | 35.5 |
8/4 Wednesday
Greg's Notebook
- Performed restriction digest of pTFc, J23100, J23106, J23109, F2620, pSB2k3, pBAD*, pSB1k3
- *There wasn't enough pBAD, so I used all that was left (~20uL). Even if this digest fails I still have the previously-digested stuff at a low-ish concentration
Laura's Notebook
NanoDrop data for Alex's miniprep samples:
| 260/280 | 260/230 | ng/uL | |
| I13500 | 1.82 | 1.31 | 221.7 |
| J23106 | 1.85 | 1.42 | 187.6 |
| J23109 | 1.88 | 1.47 210.5 |
Suggestions from Christina:
- diagnostic gels from yesterday's digestions (done by Francisco)
run promoters (pBAD, pLUX) on 0.8%, RSID's (1 and 2) and sRNA's (1, 2, 1C, 2C) on 2% gel extract everything (on separate gels), depending on how complete digestions look- if PCR cleanup, SIP treat
- PCR cleanup inserts (PCR assembled parts)
use MiniElute columns use 10 mM Tris instead of H2O (water here is acidic, and elution is pH-dependant) let sit 1 minute before eluting load 1 uL on diagnostic gel
- check enzymes to see if getting low: order more if necessary
- colony PCR
primers: complementary to prefix and suffix or VF and VR? (VF and VR- further from ends to allow subsequent sequencing)
- expected lengths with and without insert: ladder choice, gel %, extension time
10 uL reactions, run 5 uL on gel setting up reactions:
- make, aliquot master mix (SuperMix + primers)
- pick colony with innoculating needle: inoculate PCR reaction, then streak on small square or wedge on a plate (make sure plates are dry)
- screen (PCR) 5 colonies to start, but streak out 10-15 extras just in case/for further screenings
if no bands on gel, could be: not enough cells, not correct plasmid if positive results, get sequenced
