Team:Stanford/Notebook/Lab Work/Week 6

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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

8/5 Thursday

8/6 Friday