Team:Stanford/Notebook/Lab Work/Week 6

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Contents

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


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 of top wells: RSID1, RSID2, sRNA1, 100 bp ladder, sRNA2, sRNA1C, sRNA2C, 100 bp ladder, pBAD, 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

Laura's Notebook

Worked with Francisco to plan, set up ligations- From yesterday's gel, these were my approximate concentrations (3 uL loaded each lane, so band ng divided by 3 for ng/uL):

  • RSID1: 100 ng/uL
  • RSID2: 130 ng/uL
  • sRNA1: 13 ng/uL
  • sRNA2: 17 ng/uL
  • sRNA1C: 23 ng/uL
  • sRNA2C: 23 ng/uL
  • pBAD: barely visible- will redo miniprep, digestion, etc. before ligating
  • pLUX: 20 ng/uL

Ligation Recipes:

pLUX-RSID2 pLUX-sRNA1 pLUX-sRNA1C
H2O (uL) 7.0 1.0 4.0
vector: pLUX (uL) 8.0 8.0 8.0
insert (uL) 2.0 8.0 5.0
10X ligase buffer (uL) 2.0 2.0 2.0
T4 ligase (uL) 1.0 1.0 1.0


  • 2 hours at room temperature
  • transformed 0.5 uL of each ligation into 20 uL of DH10B electrocompetent cells