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)

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