Team:Stanford/Notebook/Lab Work/Week 6

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)

• 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:

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:

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

Plan for Colony PCR:

• primers: VR (~175bases from insertion site), VF (~140 bases from insertion site)
• insert lengths: GFP ~720 bp, RFP ~ 680 bp
• expected lengths on gel: without insert- 355 bp, with GFP- 1075 bp, with RFP- 1035 bp
• include positive control for PCR (from Chris- PCR reaction that has worked with this PCR mix)

Set up Colony PCR reactions (with Francisco): 0.5 uL each primer (VF and VR stocks from Alex) 9 uL PCR SuperMix

• enough for 11 reactions made, aliquot 10 uL to each of 10 PCR tubes
• pick 5 colonies each GFP-term and RFP-term- inoculate PCR reaction, then spread on small square of Kanamycin plate
• 15 extra colonies of each spread on plates

8/5 Thursday

Laura's Notebook

• 8:45am: put Chris' liquid cultures (8) in 4oC
• helped Greg with minipreps

10:00 am: meeting with Saum (IISME peer coach)

10:45 am: check in meeting with team- discussed plan for today (me: pour 2% gel, run colony PCR reactions from yesterday)

11:00 am: RET interview meeting (IISME stipend grant requirement)

• did gel purification with Francisco of pBAD, pLUX (I did pLUX, he did pBAD)

2% gel for colony PCR diagnostic- 95V, 30 minutes

• Francisco loaded other samples in top wells (PCR cleanup products, gel purification products from pBAD and pLUX)
• order (lower wells): GFP-term 1-5 (5uL of 10uL PCR rxns from yesterday + 1 uL loading dye), 100 bp ladder, 1kb ladder, RFP-term 1-5

3:35 pm: off to IISME End of Summer Celebration...

8/6 Friday