Team:Stanford/Notebook/Lab Work/Week 6

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8/2 Monday

Francisco's Notebook

GFP, RFP - terminator ligations

Gel Extraction of Term, RFP, GFP

• Used two tubes for GFP, RFP inserts. Elution volume per tube: 40 uL. Combined tubes.
• Concentrated GFP, RFP inserts in vacu-fuge. Evaporated till 20 uL left.

! Tip: Chop up gel pieces before putting in tube ! Tip: Use heat blocks filled with water to dissolve ! Tip: Tape combs together to get a larger well for gel extraction

Diagnostic Gel of Gel Extraction

• Concentrated GFP, RFP gel extracts using Vacu-fuge, evaporated 80 uL of gel extract to 20 uL.
• Loaded 3 uL of DNA sample.
  • Estimated concentration: GFP, RFP = 15 ng/uL

Ligation of GFP, RFP to linearized terminator

• Want: 150 ng of vector, 5 to 1 molar ratio of insert to vector. Resulting recipe:
  • 6 uL vector (terminator B1006)
  • 11 uL insert (GFP or RFP)
  • 2 uL ligase buffer
  • 1 uL ligase
  • total: 20 uL reaction volume
  • start: late afternoon, went overnight.

Promoter - RSID, sRNA ligations

• Inoculated pBad
• Two glass tubes, each with 6 mL LB

PCR assembly of RSID, sRNA (program FLK)

• 95 for 2 min, 94 for 30 s, 55 for 30s, 72 for 30s, (cycle last three steps 9 more times), 72 for 10 min, 15 for ever.

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

Karina's Notebook

Goals: Laura and Francisco will miniprep pBad. I'll make 2% diagnostic gel to use for the day.

Gel
Recipe: 1 g agarose, 50 mL TAE, 5 uL EtBr
Order of Ladder: 1) RSID 1
2) RSID 2
3) sRNA 1
4) 100 bp ladder
5) sRNA 2
6) sRNA 1C
7) sRNA 2C
8) 1 kb ladder
9) miniprepped pBad

Francisco has image of gel.

• can't see PCR product. Will load 5 uL of sample + 1 uL dye and run again.
  
  

Help Chris
Chris is making electrocompetent cells. Helped him get OD readings.

Sample	Reading 1	Reading 2	Reading 3
BW	.25	.40	.97
DH10B #1	.38	.53	.60
DH10B #2	.34	.47	.55

PCR Cleanup
Cleaned up PCR products using MinElute PCR purification kit. Protocol found [http://biotech.wku.edu/online_manual/MinElute%20PCR%20Purification%20Kit.pdf here].

8/3 Tuesday

Francisco's Notebook

GFP, RFP - terminator ligations

Transformed ligations

• RFP-term: time constant 4.7ms, 1787 volts
• GFP-term: 4.9, 1786 volts
• Incubated in SOC for 40 minutes, spun down at 5000 g for 3 minutes, removed 750 uL of supernatant, plated the remaining 250 uL.

• Edit: Colonies!!!

Promoter - RSID, sRNA ligations

• Miniprep pBad
• Eluted two 50 uL tubes, combined into one 100 uL tube.
• Spin columns had no residual liquid after elution

Diagnostic Gel 1 of RSID’s, sRNA’s and pBad

• Loaded 1 uL of samples
• RSID and sRNA bands were not that visible, did not have 1 kb ladder for pBad

Made 600 uL of 100 bp ladder

• 60 uL 100 bp ladder (want to load 0.5 ug of DNA with 10 uL of ladder)
• 100 uL 6x gel loading dye
• 440 uL water

Diagnostic Gels, run 2

• For PCR products:
  • Loaded 5 uL samples in 0.8% gel , RSID on top tow, sRNA on bottom row
  • Loaded bottom row during this second run - ethidium bromide may have leaked out
  • RSID bands looked good, sRNA bandswere not visible (possibly due to low EtBr)
• For pBad
  • Loaded 1 uL DNA in 2.0% gel
  • Band was visible and in the right place

Diagnostic Gel, run 3

• Karina and Greg PCR cleaned-up the PCR products
• Loaded 5 uL RSID’s and sRNA’s in 2% gel
• Bands were visible and in the right place

Digestions of promoters and PCR products

• pBad, pLux will be cut at S, P
• 34 uL DNA, 5 uL BSA, 5 uL NEBuffer 2, 3.5 uL PstI, 3.0 uL SpeI*

RSID’s, sRNA’s will be cut at X, P

• 34 uL DNA, 5 uL BSA, 5 uL NEBuffer 3, 3.5 uL XbaI, 3.0 uL PstI*
• First enzyme added in the evening, leave overnight. Heat inactivate next morning and take 1 uL sample for subsequent diagnostic gel.
• Edit: Second enzyme added the next morning, after heat inactivation.

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

Francisco's Notebook

GFP, RFP - terminator ligations

Colony PCR of RFP-term and GFP-term ligations

• Primers (check plasmid): VF2, VR
• Picked 5 colonies to PCR, streaked them on a plate (divided into a grid)
• Streaked 20+ more colonies just in case
• Positive band = ~ 1000 bp, Negative band = ~ 300 bp

• 9 uL PCR Supermix, 0.5 uL VR, 0.5 uL VF2 (unknown concentrations)
• total: 10 uL reaction volume
• Tubes labelled G1 thru G5, R1 thru R5

Promoter - RSID, sRNA ligations

• Digestions of promoters and PCR products
• Heat inactivated the overnight digestion and added the second enzyme.
• Stopped second digestion around 5 pm

Diagnostic Gels of Digestions

• Ran RSID’s and sRNA’s on 2% gel, 1 uL samples
• Ran pBad and pLux on 0.8% gel, 1 uL samples
• All bands visible, pBad was very faint

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

Francisco's Notebook

GFP, RFP - terminator ligations

Diagnostic Gel of Colony PCR’s

• Ran at 95 V for 30 minutes
• G1, G2, G3, G4, G5 all had positive bands
• R1, R3, R5 had positive bands, R4 had a very faint correct band, R2 had a negative band

Inoculated G1, G3, G5, R1, R3, R5

Promoter - RSID, sRNA ligations

• Gel Extraction of Digested pBad and pLux
• Gel run at 75V for 50 minutes
• Much of the pBad sample was stuck in the well. (Genomic DNA? But diagnostic gel of miniprep DNA had the correct band, without getting stuck in the well).
• Eluted in 30 uL EB (elution buffer = 10 mM Tris-CL pH 8.5)

PCR clean-up of digested PCR products (Karina)

• Used MinElute, eluted in 10 mM Tris-CL after letting it stand for a minute
• Used vacufuge to concentrate, 60 deg C, final volume around 15 uL

! Tip: Remember to open tube lids when using vacufuge so liquid can evaporate

Diagnostic Gel of Gel Extracts, PCR Clean-ups, and Colony PCR’s

• Ran at 95 V for 30 minutes
• Loaded 3 uL of RSID’s, sRNA’s and promoters

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...

Karina's Notebook

Goal: Run PCR cleanup of RSIDs and sRNAs. Use minElute columns (Check to see if we have them). If not, run the clean up and concentrate down to 20 uL using Speed Vac. Elute in 50 uL Tris-HCl.

MinElute columns have purple "lining" of filter, NOT white, and are stored in the 4ºC.
Don't have any, so run clean up and then use Speed Vac to concentrate.
DON'T MIX UP BUFFERS.
Digestion volume: 50 uL
Let Tris-HCl stand for 1 min before spin

Dreamweaver Workshop!
Left lab from 1:00-4:30 to learn how to update the website.

8/6 Friday

Francisco's Notebook

GFP, RFP - terminator ligations

Miniprepped G1, G3, G5, R1, R3, R5

• Colonies with successful ligations
• Used Promega Kit, eluted in 50 uL of the provided nuclease-free water
• No water left in column after elution
• Nanodrop results:
  • (Colony: 260/280, 260/230, ng/uL)
  • G1: 1.86, 1.18, 113.8
  • G3: 1.81, 1.22, 100.5
  • G5: 1.81, 1.14, 132.3
  • R1: 1.85, 1.32, 111.2
  • R3: 1.90, 1.41, 116.6
  • R5: 1.88, 1.34, 133.4

! Tip: Must elute in water if sending in DNA for sequencing

Sent in G1, G3, R1, R3 for sequencing

Promoter - RSID, sRNA ligations

• Ligations with pLux
• Did not attempt any ligations with pBad (low concentration). Started with the pLux ligations first.
• Roughly aimed for a 10:1 insert to vector molar ratio.
• Split the vector (pLux) DNA evenly between the three ligations.

All ligations: 2 uL 10x T4 ligase buffer, 1 uL T4 ligase

• pLux + RSID2: 8 uL vector, 2 uL insert, 7 uL water
• pLux + sRNA1: 8 uL vector, 8 uL insert, 1 uL water
• pLux + sRNA1c: 8 uL vector, 5 uL insert, 4 uL water

Transform pLux ligations

• Used 0.5 uL DNA. Zapper settings: 1800 V, 25 uF, 2000 ohms.
• pLux + RSID2: time constant 4.6 ms, 1786 volts
• pLux + sRNA1: 4.8, 1787 volts
• pLux + sRNA1c: 4.6, 1786 volts
• Plated overnight.
• Edit: only one colony from the pLux + sRNA 1c ligation =(

Ryan’s advice:

• Reduce volume of digestions to 10 uL.
• Don’t ligate if DNA concentration is less than 15-20 ng/uL, if there is less than 100-150 ng of vector in a 10 uL ligation, concentrate the DNA first.

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