Contents 1 June 2010 1.1 June 1/2010 1.2 June 2/2010 1.3 June 2/2010 - Evening 1.4 June 3/2010 1.5 June 3/2010 - Evening 1.6 June 5/2010 1.7 June 6/2010 1.8 June 7/2010 1.9 June 7/2010 - Evening 1.10 June 8/2010 1.11 June 8/2010 - Evening 1.12 June 9/2010 1.13 June 10/2010 1.14 June 14/2010 Evening 1.15 June 15/2010 1.16 June 15/2010 Evening 1.17 June 16/2010 1.18 June 16/2010 Evening 1.19 June 17/2010 1.20 June 17/2010 Evening 1.21 June 18/2010 1.22 June 21/2010 1.23 June 22/2010 1.24 June 22/2010 Evening 1.25 June 23/2010 1.26 June 24/2010 1.27 June 28/2010 1.28 June 30/2010

June 2010

June 1/2010

JV quantified the amount of DNA in gels run to date using ImageJ software. Results to be posted in working plasmids box.

Objective: Transform plasmids into DH5α
Method: Follow competent cell transformation protocol to transform the following:
From our ligations:

• pLacI-sRBS-Lumazine-dT
  
• pLacI-sRBS-Lumazine-dT
  
• mms6 (A6)
  
• mms6 (B6)
  
• xylE (C4)
  
• xylE (B4)
  

From the 2010 Parts Distribution:

• ECFP (Bba_E0020)
  
• EYFP (Bba_E0030)
  
• BglII Endonuclease (Bba_K112106)
  

June 2/2010

(In Lab: JV)

Objective: Isolate plasmid DNA of RBS-xylE (BBa_J33204) from DH5α cells and confirm results.

Method: "Mini-prep" the plasmid DNA using boiling lysis miniprep. Then restrict the DNA once and run on a 1% agarose gel (TAE).

Restriction Reaction

Ingredient	Volume(µL)
MilliQ H20 Water	15.75
Orange Buffer (10x)	2
pDNA (rbs-xylE)	2
EcoRI	0.25

Unrestricted Control

Ingredient	Volume(µL)
MilliQ H20 Water	16
Orange Buffer (10x)	2
pDNA (rbs-xylE)	2

DNA was restricted for 80 minutes at 37^oC.

Analyzed results on a 1% agarose gel. Load order as follows:

Lane	Sample	Volume Sample (µL)	Volume Loading Dye (µL)
1	Restricted RBS-xylE	10	2
1	Unestricted RBS-xylE†	1	2
1	1kb Ladder††	2	2

† Added 9µL MilliQ H₂O
†† Added 8µL MilliQ H₂O
Ran gel at 100V from 2 hours.
Results:

Conclusions: Plasmid DNA prep and restriction was successful.

Objective: Ligate rbs-xylE (Bba_J33204) to our double terminator, and insert it into the pSB1T3 plasmid backbone.
Method:

• Restrictions
  
  • Restrict rbs-xylE wit EcoRI and SpeI (Red Buffer)
    
  • Restrict the double terminator with XbaI and PstI (Tango Buffer)
    
  • Restrict pSB1T3 with EcoRI and PstI (Red Buffer)
    
  Set up reactions as follows:
  

  Component	Volume (µL)
  MilliQ H2O	15.5
  Buffer	2
  pDNA	2
  Enzyme	0.25 + 0.25

  Set up control reaction as follows:

  • MilliQ H₂O - 16µL
    
  • Buffer - 2µL
    
  • pDNA - 2µL
    

  Incubated reactions for 65 minutes at 37^oC
  Killed enzymes by incubating reactions for 10 minutes at 65^oC
  

• Ligation
  Reaction set up as follows:
  • T4 DNA ligase - 0.25µL
    
  • rbs-xylE - 5µL
    
  • dT - 3µL
    
  • pSB1T3 - 8µL
    
  • 10x Ligation Buffer - 2µL
    
  • MilliQ H₂O - 1.75µL
    
  Incubated reactions overnight at room temperature (total of 19.5 hours)
  Killed enzymes by incubating reactions for 10 minutes at 80^oC</ul>

  June 2/2010 - Evening

  Objective: Set up new ligations of pLacI and sRBS-Lum-dT according to Tom Knight's protocol. Previous ligation had very little DNA.
  Relevant Information:
  

  • Want a final mass of 25ng of each pDNA in the ligation mix.
  • Final concentration of pDNA in restriction digest should be 25-50ng/µL.
  • Tom Knight's restriction reaction is 50µL, therefore there should be 1000ng pDNA in each restriction digest.
  • Identified the following plasmids in our working plasmids box:

  Common Name	Location	Concentration (ng/µL)	Volume/rxn (µL)
  pLacI Maxiprep	A9	990	~1
  pLacI (B1)	A6	440	~2
  sRBS-Lum-dT (2)	A1	965	~1
  sRBS-Lum-dT (1)	A2	1145	~1
  sRBS-Lum-dT Maxiprep	B8	4780	~.2
  sRBS-Lum-dT	B7	4375	~.25
  sRBS-Lum-dT (1)	G2	335	~3
  sRBS-Lum-dT (2)	G3	965	~2

  • Make a 1:10 dilution of sRBS-Lum-dt maxiprep (D8) and sRBS-Lum-dT (B7). 0.5µL pDNA in 4.5µL water.
  • Cut pLacI with EcoRI and SpeI
  • Cut sRBS-Lum-dT with XbaI and PstI
  • Cut pSB1T3 with EcoRI and PstI
  • Will have total of 12 ligation reactions, want 12x2µL of pSB1T3 to add to each, therefore want 25µL of pSB1T3.

  Method:
  Restriction
  

  Name	[pDNA] (ng/µL)	Volume pDNA (µL)	Volume Water (µL)	Volume Buffer (µL)	Enzymes	Total Volume
  sRBS-Lum-dT (A1)	965	1	43.5	5	0.25µL XbaI 0.25µL PstI	50
  sRBS-Lum-dT (A2)	1145	1	43.5	5	0.25µL XbaI 0.25µL PstI	50
  pLacI Maxiprep (A1)	990	1	43.5	5	0.25µL EcoRI 0.25µL SpeI	50
  sRBS-Lum-dT Maxiprep(B8)	4780	2 (of 1:10 dilution)	42.5	5	0.25µL XbaI 0.25µL PstI	50
  sRBS-Lum-dT (B7)	4375	2.5 (of 1:10 dilution)	42	5	0.25µL XbaI 0.25µL PstI	50
  pLacI (D6)	440	2	42.5	5	0.25µL EcoRI 0.25µL SpeI	50
  sRBS-Lum-dT (G2)	335	3	41.5	5	0.25µL XbaI 0.25µL PstI	50
  sRBS-Lum-dT (G3)	540	2	42.5	5	0.25µL XbaI 0.25µL PstI	50
  pSB1T3	25	12.5	7	5	0.25µL EcoRI 0.25µL PstI	50

  Incubate for 30 minutes at 37^oC (Start- 12:10pm; End- 12:40pm)
  Heat kill enzymes at 80^oC for 20 minutes
  

  Ligation:
  In a 10µL final volume, add:

  • 2µL of sRBS-Lum-dT component
  • 2µL of pLacI component
  • 2µL of pSB1T3 component
  • 1µL of T4 Buffer
  • 0.25µL of T4 DNA Ligase
  • 2.75µL of MilliQ H₂O

  Incubate for 30 minutes at room temperature to ligate
  Incubate for 20 minutes at 80^oC to heat kill
  

  June 3/2010

  Carried out protocol described in June 2/2010 - Evening
  Analyzed results on 1% agarose gel.Load order as follows:
  

  Lane	Gel 1 Sample	Gel 1 Load	Gel 2 Sample	Gel 2 Load
  1	1kb Ladder	2µL dye, 2µL ladder 8µL MilliQ H2O	1kb Ladder	2µL dye, 2µL ladder 8µL MilliQ H2O
  2	Restricted sRBS-Lum-dT (A1)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(A1)	10µL DNA 2µL Dye
  3	Unrestricted sRBS-Lum-dT (A1)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(A1)	10µL DNA 2µL Dye
  4	Restricted sRBS-Lum-dT (A2)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(A2)	10µL DNA 2µL Dye
  5	Unrestricted sRBS-Lum-dT (A2)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(A2)	10µL DNA 2µL Dye
  6	Restricted sRBS-Lum-dT (B8)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(B7)	10µL DNA 2µL Dye
  7	Unrestricted sRBS-Lum-dT (B8)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(B7)	10µL DNA 2µL Dye
  8	Restricted sRBS-Lum-dT (B7)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(G2)	10µL DNA 2µL Dye
  9	Unrestricted sRBS-Lum-dT (B7)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(G2)	10µL DNA 2µL Dye
  10	Restricted sRBS-Lum-dT (G2)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(G3)	10µL DNA 2µL Dye
  11	Unrestricted sRBS-Lum-dT (G2)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(G3)	10µL DNA 2µL Dye
  12	Restricted sRBS-Lum-dT (G3)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(D6)+sRBS-Lum-dT(B8)	10µL DNA 2µL Dye
  13	Unrestricted sRBS-Lum-dT (G3)	10µL DNA 2µL Dye	pSB1T3 Ligation of: pLacI(A9)+sRBS-Lum-dT(B8)	10µL DNA 2µL Dye
  14	Restricted pLacI (A9)	10µL DNA 2µL Dye	Restricted rbs-xylE	10µL DNA 2µL Dye
  15	Unrestricted pLacI (A9)	10µL DNA 2µL Dye	Unrestricted rbs-xylE	10µL DNA 2µL Dye
  16	Restricted pLacI B1 (D6)	10µL DNA 2µL Dye	Restricted pSB1T3	10µL DNA 2µL Dye
  17	Unrestricted pLacI B1 (D6)	10µL DNA 2µL Dye	Unrestricted pSB1T3	10µL DNA 2µL Dye
  18	Unrestricted dT	10µL DNA 2µL Dye	pSB1T3 Ligation of: rbs-xylE+dT	10µL DNA 2µL Dye
  19	Restricted dT	10µL DNA 2µL Dye

  Ran gel at 100V for 90 minutes.
  Results:
  

  
  
  

  Conclusions:
  

  
  

  June 3/2010 - Evening

  Objective: Repeat restriction of pSB1T3 and ligate with pLacI and sRBS-Lum-dT. Previous ligations all used up on gel.
  Method:
  Ligation:
  In a 10µL final volume, add:

  • 2µL of sRBS-Lum-dT component
  • 2µL of pLacI component
  • 2µL of pSB1T3 component
  • 1µL of T4 Buffer
  • 0.25µL of T4 DNA Ligase
  • 2.75µL of MilliQ H₂O

  Incubate for 30 minutes at room temperature to ligate
  Incubate for 20 minutes at 80^oC to heat kill
  Following ligation, transformed using transformation protocol. Plates incubated in 37^oC incubator for 44 hours.
  Results: Only plate pLacI (D6) + sRBS-Lum-dT (G2) + pSB1T3 grew; had 2 colonies. Control plate did not grow, acidentally plated on tetracycline plate instead of ampicillin (pUC19).
  Follow-up: Inoculated 5mL LB media (tetracycline positive) with cells from the transformation plates and incubated at 37^oC overnight. (June 5/2010).
  
  

  Objective: Ligate pBad-TetR part with fluorescent protein part in pSB1C3 backbone.
  Method:
  Restriction
  

  Name	Volume pDNA (µL)	Volume Water (µL)	Volume Buffer (µL)	Enzymes	Total Volume
  pSB-NEYFP (B4)	.8	43.7	5	0.25µL XbaI 0.25µL PstI	50
  pSB-CEYFP (B5)	.9	43.6	5	0.25µL XbaI 0.25µL PstI	50
  pBad-TetR)	.3	44.2	5	0.25µL EcoRI 0.25µL SpeI	50
  NEYFP (E1)	4.3	40.7	5	0.25µL XbaI 0.25µL PstI	50
  NEYFP (E2)	0.3	42.2	5	0.25µL XbaI 0.25µL PstI	50
  Fusion CEYFP (E3)	3.9	40.6	5	0.25µL XbaI 0.25µL PstI	50
  Fusion CEYFP (E4)	2.0	42.5	5	0.25µL XbaI 0.25µL PstI	50
  Fusion CEYFP (E5)	3.0	41.5	5	0.25µL XbaI 0.25µL PstI	50
  CEYFP (E6)	0.6	43.9	5	0.25µL XbaI 0.25µL PstI	50
  CEYFP (E7)	0.5	44.0	5	0.25µL XbaI 0.25µL PstI	50
  pBad-TetR (F4)	2.5	42	5	0.25µL EcoRI 0.25µL SpeI	50
  pBad-TetR (F5)	1.7	42.8	5	0.25µL EcoRI 0.25µL SpeI	50
  pSB-CEYFP (G4)	2.9	41.6	5	0.25µL XbaI 0.25µL PstI	50
  pSB1C3	15.5	46		0.25µL EcoRI 0.25µL PstI	62

  Incubated at 37^oC for 75 minutes.
  

  • Used Red buffer for the EcoRI/SpeI and EcoRI/PstI digests
  • Used Tango buffer for the XbaI/PstI digests
  • Did not heat kill upon removal from incubation, put directly into -20^oC fridge.

  Continue Ligation on Saturday (See below).
  

  June 5/2010

  (In the lab:AS)
  Objective: Ligate restriction products from June 3/2010.
  Relevant information:
  

  • Have 3 tubes of part 1 (pBad-TetR)
    • In ampicillin backbone
  • Have 10 tubes of part 2 (fluorescent protein - various)
    • In ampicillin backbone
  • Will have 30 combinations
  • Will use pSB1C3 as plasmid backbone
    • Used most of the pSB1T3 and want to save remainder for creating new backbone via PCR.

  Method:
  *Restriction digests were not heat killed after reactions. Freezing probably killed the restriction enzymes, but I will hea kill them at 80^oC for 20 minutes anyways prior to adding Ligase.

  • • Cool on ice for 10 minutes before adding ligase.

  Master Mix	Volume/tube (µL)	Total Volume (µL)
  DNA	6	---
  10x Buffer	1	32
  T4 DNA Ligase	.25	8
  MilliQ H2O	2.75	88

  • Add 4µL master mix to each DNA tube.
    

  Follow-up: Ligation reactions will be transformed into DH5α cells
  

  
  

  June 6/2010

  (In Lab: JV, HS)
  

  Objective:
  Isolate the following plasmid DNA from DH5α:
  

  • pLacI-sRBS-Lumazine-dT in pSB1T3 (colony 2)
    
  • pLacI-sRBS-Lumazine-dT in pSB1T3 (colony 1)
    

  Method:
  Followed boiling lysis miniprep protocol. Eluted with 10µL Milli-Q H₂O and RNase A.
  

  Notes:
  

  • Placed colony 2 in cell E10 of glycerol stocks and J6 of working plasmid box.
  • Placed colony 2 in cell F1 of glycerol stocks and J5 of working plasmid box.

  Objective:
  Transformed the following plasmid DNA into DH5α cells:
  

  • pBad-TetR-CEYFP: (F5+E6), (B10+E7), (B10+E6), (F4+E6), (F5+E4), (F4+E7)
    
  • pBad-TetR-Fusion CEYFP: (F5+E3), (B10+E4), (F4+E3), (B10+E3), (B10+E5), (F4+E4), (F5+E4), (F5+E5), (F4+E5)
    
  • pBad-TetR-pSB CEYFP: (F4+B5), (B10+G4), (F4+G4), (F4+B5), (F5+B5), (F5+G4)
    
  • pBad-TetR-NEYFP: (F5+E1), (B10+E1), (F4+E2), (F5+E2), (B10+E2), (F4+E1)
    
  • pBad-TetR-pSB NEYFP: (F5+B4), (F4+B4), (B10+B4)
    
  • Positive control -> DH5α + pSB1C3
    
  • Negative control -> DH%α + Milli-Q H₂O
    

  Method:
  Followed Competent Cell Transformation protocol and used chloramphenicol as an antibiotic. We plated all 200µL of DNA onto the plates. The plates were incubated at 37^oC from 4:30pm to 10:00am.

  Results:
  None of the plates showed any growth.

  June 7/2010

  (In Lab: JV, HB, TF, AV)
  

  Objective:
  Purification of DNA to increase ligation and transformation efficiency.
  

  Method:
  BioBasic Protocol for Purification of PCR products.
  

  DNA (from Working Plasmid Box) Purified:

  • pBAD-TetR (B10)
  • pBAD- TetR (F4)
  • pBAD-TetR (F5)
  • EYFP (B1)
  • pSB-NEYFP (B4)
  • pSB-CEYFP (B5)
  • NEYFP (E1)
  • NEYFP (E2)
  • Fusion CEYFP (E3)
  • Fusion CEYFP (E4)
  • Fusion CEYFP (E5)
  • CEYFP (E6)
  • CEYFP (E7)
  • EYFP (E8)
  • EYFP (E9)
  • EYFP (E10)
  • ECFP (F1)
  • ECFP (F2)
  • ECFP (F3)
  • EYFP (G1)
  • pSB-CEYFP (G4)

  
  Objective:
  Restrict and run 1% agarose gel of plasmids J5, J6, and pSB1T3 (June 2/10).
  

  
  Method:

  Restrictions

  Component	Volume (µL)
  Restriction Enzyme (XbaI)	0.25
  Buffer (Tango)	2
  Plasmid DNA	2
  MilliQ H2O	15.75

  Incubated for 1 hour in 37^oC incubator.
  Heat shocked for 10 min at 65^oC on heating block.

  
  1% Agarose Gel

  Lane	Sample	Load (µL)
  1	1kb ladder	2 ladder + 2 dye (6X) + 8 MilliQ H2O
  2	J5-pLacI-SRBS-Lumazine-dT(1)	10 DNA + 2 Dye (6X)
  3	J5-pLacI-SRBS-Lumazine-dT(1)restricted	10 DNA + 2 Dye (6X)
  4	J6-pLacI-SRBS-Lumazine-dT(2)	10 DNA + 2 Dye (6X)
  5	J5-pLacI-SRBS-Lumazine-dT(2)restricted	10 DNA + 2 Dye (6X)
  6	pSB1T3	10 DNA + 2 Dye (6X)
  7	pSB1T3 restricted	10 DNA + 2 Dye (6X)
  8	Empty
  9	Empty
  10	Empty

  Ran at 100V for 72 min.
  

  IMAGE TO COME!!!!!!
  

  June 7/2010 - Evening

  (In Lab: AS, KG)
  

  Objective:
  Ligation of:
  

  • pBAD-TetR (F5) + CEYFP (E6)
  • pBAD-TetR (F5) + NEYFP (E1)
  • pBAD-TetR (F5) + pSB-NEYFP (B4)
  • pBAD-TetR (F5) + Fusion-CEYFP (E7)
  • pBAD-TetR (F5) + Fusion-CEYFP (E3)
  • pBAD-TetR (F5) + CEYFP (E7)
  • pBAD-TetR (F5) + NEYFP (E2)
  • pBAD-TetR (F5) + pSB-CEYFP (B5)
  • pBAD-TetR (F5) + Fusion-CEYFP (E5)
  • pBAD-TetR (F5) + pSB-CEYFP (G4)
  • pBAD-TetR (F4) + CEYFP (E7)
  • pBAD-TetR (F4) + NEYFP (E1)
  • pBAD-TetR (F4) + pSB-NEYFP (B4)
  • pBAD-TetR (F4) + pSB-CEYFP (B5)
  • pBAD-TetR (F4) + CEYFP (E6)
  • pBAD-TetR (F4) + Fusion-CEYFP (E4)
  • pBAD-TetR (F4) + Fusion-CEYFP (E3)
  • pBAD-TetR (F4) + pSB-CEYFP (G4)
  • pBAD-TetR (F4) + Fusion-CEYFP (E5)
  • pBAD-TetR (F4) + NEYFP (E2)
  • pBAD-TetR (B10) + pSB-NEYFP (B4)
  • pBAD-TetR (B10) + pSB-CEYFP (G4)
  • pBAD-TetR (B10) + NEYFP (E1)
  • pBAD-TetR (B10) + CEYFP (E6)
  • pBAD-TetR (B10) + CEYFP (E7)
  • pBAD-TetR (B10) + Fusion-CEYFP (E4)
  • pBAD-TetR (B10) + pSB-CEYFP (B5)
  • pBAD-TetR (B10) + Fusion-CEYFP (E5)
  • pBAD-TetR (B10) + Fusion-CEYFP (E3)
  • pBAD-TetR (B10) + NEYFP (E2)

  Method:
  FILL ME IN!!!!!!
  

  June 8/2010

  (In the lab: JV, AV)
  Objective: Follow the overexpression of our pLacI-sRBS-Lum-dT construct.
  Method: FILL ME OUT!!!!!!
  Results:

  Time	OD600 F1	OD600 E10
  0	0.118	0.103
  30	0.133	0.111
  30	0.145	0.116
  90
  120	0.160	0.124
  150	0.120	0.093
  180	0.129	0.100
  210	0.145	0.122
  240	0.158	0.145
  270	0.171	0.178
  300	0.194	0.222
  330	0.223	0.280
  360	0.252	0.364
  390	0.296	0.458
  420	0.338	0.557†
  450	0.394	0.656
  480	0.453	0.675
  510	0.530	0.688
  540	0.598†	0.706
  600	0.633	0.752
  660	0.653
  720	0.679
  ∞	1.278

  † Overexpression induced by adding 1mM IPTG.
  Following overexpression, 1mL of cells was removed from the culture, spun down at ~13000xg for 20 seconds, excess media removed and rinsed with water.
  Suspended cells in 8M urea, mixed with 6x dye and ran on 18% SDS-PAGE gel for 90 minutes at 200V. Gel stained overnight.
  Results: IMAGE TO COME!!!!
  
  

  Objective: Calculate quantity of DNA in pBad-TetR and fluorescent protein mini-preps by staining an agarose gel.
  Method: Restrict plasmid DNA (done by AV,HB,TF on June 7/2010) and run on a 1% TAE agarose gel (JV).
  

  Lane	Gel 1 Sample	Gel 1 Load	Gel 2 Sample	Gel 2 Load
  1	1kb Ladder	2µL dye, 2µL ladder 8µL MilliQ H2O	1kb Ladder	2µL dye, 2µL ladder 8µL MilliQ H2O
  2	Restricted EYFP (B1)	10µL DNA 2µL Dye	Restricted Fusion CEYFP (E3)	10µL DNA 2µL Dye
  3	Unrestricted EYFP (B1)	10µL DNA 2µL Dye	Unrestricted Fusion CEYFP (E3)	10µL DNA 2µL Dye
  4	Restricted pSB-CEYFP (B5)	10µL DNA 2µL Dye	Restricted Fusion CEYFP (E4)	10µL DNA 2µL Dye
  5	Unrestricted pSB-CEYFP (B5)	10µL DNA 2µL Dye	Unrestricted Fusion CEYFP (E4)	10µL DNA 2µL Dye
  6	Restricted ECFP (F2)	10µL DNA 2µL Dye	Restricted EYFP (E10)	10µL DNA 2µL Dye
  7	Unrestricted ECFP (F2)	10µL DNA 2µL Dye	Unrestricted EYFP (E10)	10µL DNA 2µL Dye
  8	Restricted pSB-CEYFP (G4)	10µL DNA 2µL Dye	Restricted pSB NEYFP (B4)	10µL DNA 2µL Dye
  9	Unrestricted pSB-CEYFP (G4)	10µL DNA 2µL Dye	Unrestricted pSB NEYFP (B4)	10µL DNA 2µL Dye
  10	Restricted EYFP (G1)	10µL DNA 2µL Dye	Restricted ECFP (F3)	10µL DNA 2µL Dye
  11	Unrestricted EYFP (G1)	10µL DNA 2µL Dye	Unrestricted ECFP (F3)	10µL DNA 2µL Dye
  12	Restricted NEYFP (E2)	10µL DNA 2µL Dye	pBad-TetR (F5)	10µL DNA 2µL Dye
  13	Unrestricted NEYFP (E2)	10µL DNA 2µL Dye	Restricted Fusion CEYFP (E5)	10µL DNA 2µL Dye
  14	Restricted pBad-TetR (B10)	10µL DNA 2µL Dye	Unrestricted Fusion CEYFP (E5)	10µL DNA 2µL Dye
  15	Unrestricted pBad-TetR (B10)	10µL DNA 2µL Dye	pBad-TetR (F4)	10µL DNA 2µL Dye
  16	Restricted CEYFP (E6)	10µL DNA 2µL Dye	Restricted pSB1T3	10µL DNA 2µL Dye
  17	Unrestricted CEYFP (E6)	10µL DNA 2µL Dye	Unrestricted pSB1T3	10µL DNA 2µL Dye
  18	Restricted NEYFP (E1)	10µL DNA 2µL Dye		10µL DNA 2µL Dye
  19	Unrestricted NEYFP (E1)	10µL DNA 2µL Dye
  20	Restricted ECFP (F1)	10µL DNA 2µL Dye
  21	Unrestricted ECFP (F1)	10µL DNA 2µL Dye
  22	Restricted EYFP (E9)	10µL DNA 2µL Dye
  23	Unrestricted EYFP (E9)	10µL DNA 2µL Dye
  24	Restricted CEYFP (E7)	10µL DNA 2µL Dye
  25	Unrestricted CEYFP (E7)	10µL DNA 2µL Dye
  26	Restricted EYFP (E8)	10µL DNA 2µL Dye
  27	Unrestricted EYFP (E8)	10µL DNA 2µL Dye

  Ran gel at 100V for 90 minutes.
  Results:
  

  

  No bands visible except for pSB1T3 lanes, therefore could not quantify anything that had not already been quantified.
  Also, purification of DNA done on June 7/2010 seemed to reduce amount of pDNA in sample.
  
  

  Objective: Restrict mms6 (D9,D10) and dT (C1) so we can ligate the dT onto the mms6 coding region.
  Method:
  mms6 Restriction
  

  • 2µL mms6 pDNA
  • 2µL Red buffer
  • 0.25µL EcoRI
  • 0.25µL SpeI
  • 15.5µL MilliQ H₂O

  dT Restriction
  

  • 2µL dT pDNA
  • 2µL Orange buffer
  • 0.25µL EcoRI
  • 0.25µL XbaI
  • 15.5µL MilliQ H₂O

  Incubated for 1 hour at 37^oC
  Heat shock on heat block (80^oC) for 20 minutes
  Ligation

  • 2µL dT pDNA
  • 2µL mms6 pDNA
  • 0.25µL T4 DNA Ligase
  • 1µL 10x buffer
  • 4.75µL MilliQ H₂O

  Analyze results on 1% TAE agarose gel
  

  Lane	Sample	Load (µL)
  1	1kb ladder	0.5 ladder; 2 dye; 9.5 MilliQ H2O
  2	Unrestricted mms6 (D9)	10 DNA; 2 Dye
  3	Restricted mms6 (D9)	10 DNA; 2 Dye
  4	Unrestricted mms6 (D10)	10 DNA; 2 Dye
  5	Restricted mms6 (D10)	10 DNA; 2 Dye
  6	Unrestricted dT (C1)	10 DNA; 2 Dye
  7	Restricted dT (C1)	10 DNA; 2 Dye
  8	Empty
  9	Empty
  10	Empty

  Results:
  

  
  
  

  Looks like the mms6 DNA was not cut at all. Therefore is doubtful that the ligations will work.
  
  

  June 8/2010 - Evening

  Objective: Transform pLacI-sRBS-Lum-dT constructs assembled via three antibiotic assembly on June 3/2010. Also transform mms6-dT constructs assembled today using old insertion method.
  Method: Follow transformation protocol. Results: No colonies anywhere
  

  June 9/2010

  (in the lab: TF, JV)
  Objective: Transform mms6-dT ligation reactions from June 8/2010.
  Method: Followed transformation protocol and transformed the following:

  • mms6 (D9) + dT (C1)
  • mms6 (D10) + dT (C1)
  • pUC19 (positive control)
  • Water (negative control)

  Results: No transformants on plates.
  
  

  June 10/2010

  (In the lab: AV, HB, JV)
  Objective: Repeat ligation of mms6 (B9,D9,D10) and dT (C1).
  Method:
  mms6 Restriction
  

  • 2µL mms6 pDNA
  • 2µL Red buffer
  • 0.25µL EcoRI
  • 0.25µL SpeI
  • 15.5µL MilliQ H₂O

  dT Restriction
  

  • 2µL dT pDNA
  • 2µL Orange buffer
  • 0.25µL EcoRI
  • 0.25µL XbaI
  • 15.5µL MilliQ H₂O

  Incubated for 1 hour at 37^oC.
  Killed enzymes by heating to 80^oC for 20 minutes
  Ligation

  • 5µL dT pDNA
  • 5µL mms6 pDNA
  • 0.5µL T4 DNA Ligase
  • 2µL 10x buffer
  • 7.5µL MilliQ H₂O

  Incubated at room temperature overnight.
  Results:
  Analyzed on 1% TAE agarose gel:

  Lane	Sample	Load (µL)
  1	1kb ladder	0.5 ladder; 2 dye; 9.5 MilliQ H2O
  2	Unrestricted mms6 (B9)	5 DNA; 2 dye; 5 MilliQ H2O
  3	Restricted mms6 (B9)	5 DNA; 2 dye; 5 MilliQ H2O
  4	Unrestricted mms6 (D9)	5 DNA; 2 dye; 5 MilliQ H2O
  5	Restricted mms6 (D9)	5 DNA; 2 dye; 5 MilliQ H2O
  6	Unrestricted mms6 (D10)	5 DNA; 2 dye; 5 MilliQ H2O
  7	Restricted mms6 (D10)	5 DNA; 2 dye; 5 MilliQ H2O
  8	Unrestricted dT (C1)	5 DNA; 2 dye; 5 MilliQ H2O
  9	Restricted dT (C1)	5 DNA; 2 dye; 5 MilliQ H2O
  10	D9 + C1 Ligation (June 8/2010)	5 DNA; 2 dye; 5 MilliQ H2O

  Gel run for 80 minutes at 100V
  

  
  
  

  Looks like everything was restricted. Ligations may work this time.
  Follow-up: Ligation mixtures can be restriction tested and (if cutting works) transformed into DH5α cells.
  
  

  Objective: Transform pDNA from distribution plates into DH5α cells to generate glycerol stocks for lab.
  Method:
  Remove DNA from the following locations on the 2010 distributions kits:

  • double terminator (B0010-B0010; AKA B0017) from kit plate 2 well 6K (pSB1A2)
  • double terminator (B0010-B0012; AKA B0015) from kit plate 1 well 23L (pSB1AK3)

  Transform into DH5α cells using transformation protocol, with pUC19 as positive control (1ng/µL), and water as negative control.
  Results:
  

  • Positive control: TMTC (too many to count) colonies
  • B0010-B0010: 41 colonies
  • B0010-B0012: 120 colonies

  June 14/2010 Evening

  (In the lab: TF, DM, HS)
  Objective: Restriction Digest of RBS-xylE with EcoRI and SpeI
  

  Method:
  

  Restriction Digestions
  

  • Pipetted 15.5µL of water, into 2 reaction tubes (0.6mL) each.
  • Pipetted 2µL of red buffer into each of them.
  • Pipetted 2µL of rbs-xylE plasmid DNA into both tubes, but taking only the thawed liquid on the side.
  • Because this was not proper protocol for aliquoting DNA, we thawed the DNA out completely and then put 2µL more of the rbs-xylE plasmid DNA in.
  • Pipetted 0.25µL of EcoRI enzyme and 0.25µL of SpeI enzyme into the restriction reaction tube.
  • Placed in incubator (37^oC) for 1 hour.
  • Placed in ice for 10 minutes.

  Objective: Test Ligations of rbs-xylE with T4-Ligase from iGEM -20^oC and from HJ's lab's -20^oC.
  

  Method:
  

  • Pipette 12.75µL of Milli-Q H₂O into 4 mincrocentrifuge tubes.
  • Pipette 2µL of 10X T4-Ligase buffer into all 4 tubes.
  • Pipette 5µL of the RD product into both iGEM ligase tubes and HJ-Lab Ligase tubes.
  • Pipetted 5µL of the RD control into both iGEM ligase tubes and HJ-Lab ligase tubes.
  • Incubate overnight

  June 15/2010

  (In the lab: JV)
  

  Objective:Continue T4-Ligase check and confirm that it is functional.
  

  Method:Run plasmid DNA;uncut, cut, and ligated on a 1% agarose gel (TAE)
  

  Lane	Sample	Load (µL)
  1	rbs-xylE	10 DNA solution + 2 Dye
  2	rbs-xylE R.D.	10 DNA solution + 2 Dye2O
  3	rbs-xylE [iGEM ligation]	10 DNA solution + 2 Dye
  4	rbs-xylE [HJ ligation]	10 DNA solution + 2 Dye
  5	rbs-xylE [iGEM ligation control]	10 DNA solution + 2 Dye
  6	rbs-xylE [HJ ligation control]	10 DNA solution + 2 Dye
  7	1kb ladder	2 ladder + 2 dye + 8 Milli-Q H2O
  8	mms6 (B9) R.D.	10 DNA solution + 2 Dye
  9	mms6 (B9)	10 DNA solution + 2 Dye
  10	mms6 (D10) R.D.	10 DNA solution + 2 Dye
  11	mms6 (D10)	10 DNA solution + 2 Dye
  12	mms6 (D9) R.D.	10 DNA solution + 2 Dye
  13	mms6 (D19)	10 DNA solution + 2 Dye
  14	dt (C1) R.D.	10 DNA solution + 2 Dye
  15	dt (C1)	10 DNA solution + 2 Dye

  
  Result:Ran gel for 89 minutes at 100V. Gel was unreadable. I will redo the gel using and 8 well comb. IMAGE TO COME!!!!
  

  
  

  Lane	Sample	Load (µL)
  1	rbs-xylE R.D.	5 DNA solution + 1 Dye
  2	rbs-xylE	5 DNA solution + 1 Dye
  3	iGEM ligation	10 DNA solution + 2 Dye
  4	iGEM ligation control	10 DNA solution + 2 Dye
  5	HJ ligation	10 DNA solution + 2 Dye
  6	HJ ligation control	10 DNA solution + 2 Dye
  7	1kb ladder	2 ladder + 2 dye + 8 Milli-Q H2O

  Ran gel at 100V for 82 minutes.
  

  Result:The gel "broke" while running. Jeff hypothesized the cause to be excessive heat. However, the DNA was still visible.IMAGE TO COME!!!!
  

  Objective:Isolate plasmid DNA from DH5α cells.
  

  Method:The plasmid DNA is:
  

  • 1)double terminator B0017 (B0010-B0010) from 2010 kit plate 2:6K pSB1A2
  • 2)double terminator B0015 (B0010-B0012) from 2010 kit plate 1:23L pSB1AK2

  This will give us 2 additional dt's (3 total) into our working plasmid box to ligate onto the end of our constructs.
  

  Used the boiling lysis miniprep protocol and elute with 50µL of Milli-Q H₂O with RNAse A.
  

  These plasmids (labeled 1 & 2 above) are in:
  

  • working plasmid box: 1). J9 2). J10
    
  • glycerol sotck 2010 box: 1). F2 2). F3
    

  
  DNA was then purified using the protocol for purification of PCR products (BioBasic). DNA was eluted with 35µL of elution buffer.
  

  June 15/2010 Evening

  (In the lab: AS)
  

  Adam: not convinced that the rbs-xylE was cut properly last night. There doesn't appear to be and band where the insert should be.
  

  Objective: To confirm that EcoRI & SpeI are cutting (also PstI). Use rbs-xylE as test plasmid DNA.
  

  Method: Digest rbs-xylE with the following enzyme combinations:
  

  • EcoRI + SpeI (old [i.e. John's]) + Red Buffer
  • EcoRI + SpeI (new) + Red Buffer
  • EcoRI + PstI + Red Buffer
  • EcoRi + Red Buffer
  • SpeI (old) + Tango Buffer
  • SpeI (new) + Tango Buffer
  • PstI + Red Buffer
  • No enzyme controls (Red & Tango Buffer)

  Reaction mix as follows:
  

  • Milli-Q H₂O 15.5µL
  • 10X Buffer 2µL
  • pDNA 2µL
  • Enzymes 0.25µL + 0.25µL
    

  Incubated at 37^oC for 1 hour . Analyze on 1% TAE Agarose gel as follows:
  

  Lane	Sample	Load (µL)
  1	No Enzyme Control (Tango)	10 DNA solution + 2 Dye
  2	No Enzyme Control (Red)	10 DNA solution + 2 Dye
  3	PstI	10 DNA solution + 2 Dye
  4	SpeI (old)	10 DNA solution + 2 Dye
  5	SpeI (new)	10 DNA solution + 2 Dye
  6	EcoRI	10 DNA solution + 2 Dye
  7	EcoRI + SpeI (old)	10 DNA solution + 2 Dye
  8	EcoRI + SpeI (new)	10 DNA solution + 2 Dye
  9	EcoRI + PstI	10 DNA solution + 2 Dye
  10	1kb ladder	0.5 ladder + 2 dye + 9.5 Milli-Q H2Oe

  
  Results: Everything cuts exactly as it should. Continue with ligation and analysis. IMAGE TO COME!!!!
  

  June 16/2010

  (in lab: JV) Objective: To miniprep Adam's overnight cultures of xylE and rbs. Completing this will give us a working stock of this plasmid.
  

  Method: Use the boiling lysis miniprep protocol and pufrify using BioBasic protocol for purification of PCR products.
  

  Objective: Transform pLacI-sRBS-lumazine synthase-dt into BL21(DE3).
  

  Method:

  • Followed transformation protocol
    

  Changes to the protocol include:
  

  • Added 5µL DNA to 50µL BL21(DE3).
  • Incubated in 500µL SOC instead of 250µL LB
  • incubated for 90 minutes at 37^oC at 4:30pm.

  Results: There wasn't any growth after overnight incubation. Possibility of an antibiotic mix-up.

  June 16/2010 Evening

  (in lab: ADS)

  Objective: Continue with ligation test. Use restriction products to test T4 DNA ligase.
  

  Method: Have 10µL of DNA remaining for each restriction. Ligate together one of the single cut reactions, and ligate one of the double cut reactions. 4µL of each sample will be tested against each ligase.
  i.e.
  

  • EcoRI +SpeI (new) cut vs HJ's ligase
  • EcoRI + SpeI (new) cut vs iGEM ligase
  • SpeI (new) cut vs HJ's ligase
  • SpeI (new) cut vs iGEM ligase
    

  Reaction Mixture:
  

  • DNA -> 4µL
  • 10X Buffer -> 2µL
  • Milli-Q -> 13.5µL
  • Ligase -> 0.5µL
    

  Prior to setting up reaction, heat kill restriction enzymes by heating to 80^oC for 20 minutes and subsequently cooling on ice for 10 minutes.
  

  Ligations begun at 6:50pm
  

  Will run samples at 30 minutes reaction time and overnight.
  

  Analyze ligations in a 1% TAE Agarose gel
  

  Lane	Sample	Load (µL)
  1	No Enzyme Control (from last night)	1 DNA solution + 1 Dye + 4 H2O
  2	EcoRI + SpeI (old)(from last night)	1 DNA solution + 1 Dye + 4 H2O
  3	EcoRI + SpeI (new) vs HJ's Ligase	5 DNA solution + 1 Dye
  4	EcoRI + SpeI (new) vs iGEM ligase	5 DNA solution + 1 Dye
  5	SpeI (new) vs HJ's Ligase	5 DNA solution + 1 Dye
  6	SpeI (new) vs iGEM ligase	5 DNA solution + 1 Dye
  7	SpeI (old)(from last night)	1 DNA solution + 1 Dye + 4 H2O
  8	1kb Ladder	0.25 Ladder + 4.75 H2O + 1 Dye

  
  No observable ligation occurring 30 minutes with both ligases.
  

  Objective: Prepare DNA for sequencing
  

  Method: Need 20µL of DNA at 100ng/µL for each reaction. Need 20µL of 10µM primer for every 5 reactions.
  

  Plasmids that need to be sequenced:
  

  Name	Concentration (ng/µL)	Dilution Factor	Final Concentration (ng/µL)
  A8-CFP complete	1395	1/5	280
  B4-pSB NEYFP	1105	1/5	240
  B5-pSB CEYFP	1055	1/5	210
  B6-CFP complete	1285	1/5	260
  D7-xylE	1820	1/10	182
  D8-xylE	420	1/2	210
  E1-NEYFP	235	1/2	117.5
  E2-NEYFP	2980	1/10	300
  E3-Fusion CEYFP	255	1/2	122.5
  E4-Fusion CEYFP	490	1/2	245
  E5-Fusion CEYFP	335	1/2	335
  E6-CEYFP	1605	1/10	160
  E7-CEYFP	1930	1/10	190
  G4-pSB CEYFP	340	1/4	85
  G5-CFP complete	355	1.4	89

  Total of 15 primers -> 30 reactions
  60µL of each VF2 & VR primers (10µL) -> send 65µL

  June 17/2010

  (in lab: JV, HB) Objective:Run Agarose gel of overnight samples from June 16/10 to test T4 DNA ligase
  

  Method:Analyze ligation on a 1% TAE agarose gel.
  

  Lane	Sample	Load (µL)
  1	No Enzyme Control (from June 15)	2 DNA solution + 2 Dye + 8 H2O
  2	EcoRI + SpeI (old)(from June 15)	2 DNA solution + 2 Dye + 8 H2O
  3	EcoRI + SpeI (new) vs HJ's Ligase	10 DNA solution + 2 Dye
  4	EcoRI + SpeI (new) vs iGEM ligase	10 DNA solution + 2 Dye
  5	SpeI (new) vs HJ's Ligase	10 DNA solution + 2 Dye
  6	SpeI (new) vs iGEM ligase	10 DNA solution + 2 Dye
  7	SpeI (old)(from last night)	10 DNA solution + 2 Dye
  8	1kb Ladder	0.5 Ladder + 9.5 H2O + 2 Dye

  Ran for 80 minutes at 100V.

  Results:
  IMAGE TO COME!!!!
  

  June 17/2010 Evening

  (in lab: JS, AS, KG)
  

  Objective:Do PCR to observe ligations
  

  Method:
  

  Master Mix	Volume/tube (µL)	Total Volume (µL)
  MilliQ H2O	10.8	59.4
  DNA	2	-------
  5X Phusion Buffer	4	22
  10µM dNTP's	.25	8
  Forward Primer	1	5.5
  Reverse Primer	1	5.5
  Polymerase	0.2	1.1

  
  Reaction Mixtures:
  

  • HJ Double
  • iGEM Double
  • HJ Single
  • iGEM Single
  • controls (no forward or reverse primer and no DNA)
    

  June 18/2010

  (in lab: JV)
  

  Objective:Observe the ligations from June 17/10 that were amplified using PCR.
  

  Method:Observe using 1% TAE agarose gel electrophoresis. Ran the gel for 80 minutes at 100V.
  

  Lane	Sample	Volume (µL)
  8	1kb Ladder	2 Ladder + 8 H2O + 2 Dye
  2	control no DNA	20 PCR reaction + 4 6X Dye and used 12 of this solution
  3	control no reverse primer	20 PCR reaction + 4 6X Dye and used 12 of this solution
  4	control no forward primer	20 PCR reaction + 4 6X Dye and used 12 of this solution
  5	HJ single	20 PCR reaction + 4 6X Dye and used 12 of this solution
  6	iGEM single	20 PCR reaction + 4 6X Dye and used 12 of this solution
  7	HJ double	20 PCR reaction + 4 6X Dye and used 12 of this solution
  8	HJ double	20 PCR reaction + 4 6X Dye and used 12 of this solution

  Results:IMAGE TO COME!!!!
  
  

  June 21/2010

  (in lab: JV)
  

  Objective:Create more pSB1A3 plasmid.
  

  Method:Run a PCR of the plasmid.
  

  DIlute 10X -> 0.1 + 0.9 MilliQ H₂O DIlute 10X -> 0.1 + 0.9 MilliQ H₂O

  Master Mix	Volume/tube (µL)
  MilliQ H2O	10.8
  DNA	2
  5X Phusion Buffer	4
  10µM dNTP's	.25
  Forward Primer	1
  Reverse Primer	1
  Polymerase	0.2

  Use ligtest setting. WIll run 36 cycles.

  Use 3 controls:

  • no forward primer
  • no reverse primer
  • no DNA

  Results:PCR samples were run on a gel (1% 1XTAE) with the PCR samples from June 22/10. This gel was run on June 22/10
  

  June 22/2010

  (in lab: JV, HS)
  

  Objective:To overexpress pLacI-sRBS-lumazine synthase-dt in DH5α cells.
  

  Method:Incubate cells in 500mL LB w/Tet. At OD 0.6 (600λ) induce overexpression of lumazine synthase with IPTG (1µM). Take T_o, T₁, T₂, T₃ readings after induced with IPTG.

  Time	OD F1 (600λ)	OD E10 (600λ)
  0	0.078	0.072
  60	0.122	0.110
  90	0.128	0.125
  120	0.140	0.138
  150	0.149	0.157
  180	0.169	0.183
  210	0.205	0.230
  240	0.243	0.276
  270	0.278	0.323
  300	0.317	0.398
  330	0.394	0.428
  360	0.394	0.480
  390	0.441	0.553
  400	----	0.594 (induced)
  410	0.502	-----
  450	0.580 (induced)	0.736
  510	0.804	1.005
  570	1.065	1.066
  630	1.053	1.043
  ∞	0.055 (1/10 dilution)	0.078 (1/10 dilution)

  
  Objective:PCR amplify the following biobricks:
  

  • dt (23L)
  • dt (6K)
  • dt (C1)
  • mms6
  • control (no DNA)

  Method:
  

  Master Mix	Volume/tube (µL)	Total Volume (µL)
  MilliQ H2O	----	89.6
  DNA	2	-------
  5X Phusion Buffer	4	24
  10µM dNTP's	.4	2.4
  Forward Primer	0.2	1.2
  Reverse Primer	0.2	1.2
  Polymerase	0.2	1.2

  
  

  1.2% Agarose gel electrophoresis (1X TAE), used to analyze the above PCR's. 25mL of agarose solution was used in the small gel rig.
  

  Lane	Sample	Volume (µL)
  1	pSB1A3	1 PCR sample + 2 Dye + 9 H2O
  2	no DNA	1 PCR sample + 2 Dye + 9 H2O
  3	no reverse primer	1 PCR sample + 2 Dye + 9 H2O
  4	no forward primer	1 PCR sample + 2 Dye + 9 H2O
  5	1kb ladder	0.5 ladder + 2 dye + 9.5 Milli-Q H2O
  6	mms6	1 PCR sample + 2 Dye + 9 H2O
  7	dt (23L)	1 PCR sample + 2 Dye + 9 H2O
  8	dt (6K)	1 PCR sample + 2 Dye + 9 H2O
  9	dt (C1)	1 PCR sample + 2 Dye + 9 H2O
  10	no DNA	1 PCR sample + 2 Dye + 9 H2O

  
  

  Ran the gel for 40 minutes at 100V.
  

  June 22/2010 Evening

  (in lab: KG, AS)
  

  Objective:To run a PCR of:
  

  • dt (J9,J10,C1)
  • mms6 (B9,F10)
  • rbs-xylE (I1,I2)
  • xylE (D7, D8)
  • pLacI-sRBS-lumazine synthase-dt (I8,I9)
  • sRBS-lumazine synthase-dt (A1,A2,B7,B8,G2,G3)
  • pLacI (A9,D1)
  • pSB1A3
    

  so that we have more DNA for restrictions and ligations.
  

  
  Method:
  

  Master Mix 1	Volume/tube (µL)	Total Volume (µL)
  MilliQ H2O	10.8	226.8
  DNA	2	4
  5X Phusion HF Buffer	4	84
  10µM dNTP's	1	21
  Forward Primer (VF2)	1	21
  Reverse Primer (VR)	1	21
  Fimnzzymes polymerase	0.2	4.2

  
  

  Master Mix 2	Volume/tube (µL)	Total Volume (µL)
  MilliQ H2O	10.8	226.8
  DNA	2	4
  5X Econo Taq Buffer	4	84
  10µM dNTP's	1	21
  Forward Primer (VF2)	1	21
  Reverse Primer (VR)	1	21
  Imitrages polymerase (Econo Taq)	0.2	4.2

  
  

  Master Mix for pSB1A3	Volume/tube (µL)	Total Volume (µL)
  MilliQ H2O	10.8	31
  DNA	1	20
  5X Phusion Buffer	4	10
  10µM dNTP's	1	2.5
  Primer (SB-prep-2Ea)	1	2.5
  Primer (SB-prep-3P)	1	2.5
  Fimnzzymes polymerase	0.2	0.5

  
  

  Master Mix for pSB1A3	Volume/tube (µL)
  MilliQ H2O	10.8
  DNA	2
  5X Econo Taq Buffer	4
  10µM dNTP's	1
  Primer (SB-prep-2Ea)	1
  Primer (SB-prep-3P)	1
  Imitrages polymerase	0.2

  
  

  June 23/2010

  (in lab: JV, HS)
  

  Objective:: To run the pcr products on an agarose gel.
  

  Method: Run on 1% agarose gel using the large gel rig.
  

  Lane	Sample	Load (µL)
  1	1kb DNA Ladder	0.5 Ladder + 2 Dye + 9 H2O
  2	Phu-dt-J9	5 PCR sample + 2 Dye + 3 H2O
  3	Phu-dt-J10	5 PCR sample + 2 Dye + 3 H2O
  4	Phu-dt-C1	5 PCR sample + 2 Dye + 3 H2O
  5	Fus-mms6-B9	5 PCR sample + 2 Dye + 3 H2O
  6	Fus-mms6-F10	5 PCR sample + 2 Dye + 3 H2O
  7	Fus-rbs-xylE-I1	5 PCR sample + 2 Dye + 3 H2O
  8	Fus-rbs-xylE-I2	5 PCR sample + 2 Dye + 3 H2O
  9	Fus-xylE-D7	5 PCR sample + 2 Dye + 3 H2O
  10	Fus-xylE-D8	5 PCR sample + 2 Dye + 3 H2O
  11	Fus-pLacI-sRBS-lum-dt I8	5 PCR sample + 2 Dye + 3 H2O
  12	Fus-pLacI-sRBS-lum-dt I9	5 PCR sample + 2 Dye + 3 H2O
  13	Fus-sRBS-lum-dt A1	5 PCR sample + 2 Dye + 3 H2O
  14	Phu-sRBS-lum-dt A2	5 PCR sample + 2 Dye + 3 H2O
  15	Phu-sRBS-lum-dt B7	5 PCR sample + 2 Dye + 3 H2O
  16	Phu-sRBS-lum-dt B8	5 PCR sample + 2 Dye + 3 H2O
  17	Phu-sRBS-lum-dt G2	5 PCR sample + 2 Dye + 3 H2O
  18	Phu-sRBS-lum-dt G3	5 PCR sample + 2 Dye + 3 H2O
  19	Phu-pLacI A9	5 PCR sample + 2 Dye + 3 H2O
  20	Phu-pLacI D1	5 PCR sample + 2 Dye + 3 H2O
  21	dt J9	5 PCR sample + 2 Dye + 3 H2O
  22	dt J10	5 PCR sample + 2 Dye + 3 H2O
  23	dt C1	5 PCR sample + 2 Dye + 3 H2O
  24	mms6 B9	5 PCR sample + 2 Dye + 3 H2O
  25	mms6 F10	5 PCR sample + 2 Dye + 3 H2O
  26	rbs-xylE I1	5 PCR sample + 2 Dye + 3 H2O
  27	rbs-xylE I2	5 PCR sample + 2 Dye + 3 H2O
  28	xylE D7	5 PCR sample + 2 Dye + 3 H2O
  29	xylE D8	5 PCR sample + 2 Dye + 3 H2O
  30	pLacI-sRBS-lum-dt I8	5 PCR sample + 2 Dye + 3 H2O
  31	pLacI-sRBS-lum-dt I9	5 PCR sample + 2 Dye + 3 H2O
  32	sRBS-lum-dt A1	1 PCR sample + 2 Dye + 9 H2O
  33	sRBS-lum-dt A2	1 PCR sample + 2 Dye + 9 H2O
  34	sRBS-lum-dt B7	1 PCR sample + 2 Dye + 9 H2O
  35	sRBS-lum-dt B8	1 PCR sample + 2 Dye + 9 H2O
  36	sRBS-lum-dt G2	1 PCR sample + 2 Dye + 9 H2O
  37	sRBS-lum-dt G3	1 PCR sample + 2 Dye + 9 H2O
  38	sRBS-lum-dt A9	1 PCR sample + 2 Dye + 9 H2O
  39	sRBS-lum-dt D1	0.5 ladder + 2 dye + 9.5 Milli-Q H2O
  40	pSB1A3 control	5 PCR sample + 2 Dye + 3 H2O
  41	pSB1A3	5 PCR sample + 2 Dye + 3 H2O
  42	pSB1A3 control	5 PCR sample + 2 Dye + 3 H2O
  43	pSB1A3	5 PCR sample + 2 Dye + 3 H2O

  
  

  Ran gel for 45 minutes at 100V and stained in EtBr for 20 minutes and de-stained for 10 minutes.

  • all Econe tubes are red
  • all Phusion tubes are blue
    

  Results: IMAGE!!!

  
  Objective: Adam put in overnight cultures on June 22. I want to extract the plasmid DNA.
  

  Method:
  

  • Put in overnight cultures at 8:30pm and taken out at 9:30am next morning.
  • All cultures were DH5α in LB w/ Amp.
  • Cultures were incubated at 37_oC.
  • Next morning cultures were "minipreped" using boiling lysis miniprep protocol.
    

  
  Results: Will view plasmid DNA extracted, on a 1% agarose gel (1X TAE) run at 100V for an hour.
  

  
  

  Lane	Sample	Load (µL)
  1	1kb Ladder	0.5 Ladder + 2 Dye (6X) + 9.5 Milli-Q H2O
  2	CFP complete 2010 box C8	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  3	Fusion CEYFP 2007 box H5	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  4	NEYFP 2007 box J6	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  5	Fusion CEYFP 2007 box J5	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  6	CFP complete 2010 box A10	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  7	pSB-NEYFP 2010 box B6 (C1?)	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  8	Fusion CEYFP 2007 box I5	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  9	xylE 2007 box B9	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  10	CEYFP 2007 box G6	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  11	CFP complete 2010 box A6	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  12	pSB-CEYFP 2010 box C5	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  13	pSB-NEYFP 2010 box B6 (C1?)	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  14	pSB-CEYFP 2010 BOX C3	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  15	xylE 2007 box C4	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  16	CEYFP 2007 box H6	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  17	NEYFP 2007 box J6	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O

  
  

  Picture to come!!!!!!!!!
  

  June 24/2010

  (in lab: JV, HS, AV)
  

  Objective:: To run a PCR of biobrick parts from out working plasmid box.
  

  Method:
  

  Master Mix	Volume/tube (µL)	Total Volume (µL)
  MilliQ H2O	12.8	448
  DNA	1	-------
  5X Phusion Buffer	4	140
  10µM dNTP's	1	35
  Forward Primer	0.5	17.5
  Reverse Primer	0.5	17.5
  Polymerase	0.2	7

  
  

  Ran for 25 cycles on lig25 setting.

  Objective:: Restriction Digest, Run on agarose gel, and ligate PCR products from June 23/10.
  

  Method:
  

  Six tubes for restriction digest:
  

  Part 1	Part 2
  Lane 8 (I2)	Lane 4 (C1)
  Lane 19 (A9)	Lane 8 (I2)
  Lane 19 (A9)	Lane 8 (I2)

  
  

  Restriction Digest Reaction Mixture:
  

  Ingredient	Volume (µL)
  Plasmid DNA	2
  Enzyme	0.5
  Buffer	2
  Milli-Q H2O	15.5

  
  

  Incubate at 37^oC for 1 hour. Then heat kill the enzymes at 80^oC for 20 minutes.
  

  2% Agarose gel electrophoresis:
  

  Lane	Sample	Load (µL)
  1	50bp Ladder	1 Ladder + 2 Dye (6X) + 5 Milli-Q H2O
  2	Fus-RBS-xylE (I2); Lane 8	5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O
  3	Fus-RBS-xylE (I2) restricted; Lane 8	5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O
  4	Phu-dT (C1); Lane 4	5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O
  5	Phu-dT (C1) restricted; Lane 4	5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O
  6	Phu-pLacI (A9); Lane 19(1)	5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O
  7	Phu-pLacI (A9) restricted; Lane 19(1)	5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O
  8	Phu-pLacI (A9); Lane 19(2)	5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O
  9	Phu-pLacI (A9) restricted; Lane 19(2)	5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O
  10	Fus-RBS-xylE (I2); Lane 8(1)	5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O
  11	Fus-RBS-xylE (I2) restricted; Lane 8(1)	5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O
  12	Fus-RBS-xylE (I2); Lane 8(2)	5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O
  13	Fus-RBS-xylE (I2) restricted; Lane 8(2)	5 PCR sample + 2 Dye (6X) + 5 Milli-Q H2O

  
  

  Ran gel at 100V for 60 min and stained in EtBr for 15 min.

  Picture to come!!!!!!!!!
  

  
  Objective:: Run 1% agarose gel of PCR samples from June 24/10.
  

  Lane	Sample	Load (µL)
  1	A4-pBAD	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  2	A4-pBAD	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  3	A6-SRBS	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  4	A7-SRBS	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  5	A8-CFP Complete	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  6	A10-SRBS	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  7	B1-EYFP	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  8	B2-N-term tag	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  9	B4-pSB-NEYFP	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  10	B5-pSB-NEYFP	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  11	B6-CFP	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  12	B10-pBAD-TetR	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  13	D3	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  14	D4-C-term tag	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  15	D5-C-term tag	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  16	D6-PLacI	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  17	E2-NEYFP	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  18	E6-CEYFP	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  19	E7-CEYFP	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  20	1kb ladder	0.5 ladder + 2 Dye (6X) + 9.5 Milli-Q H2O
  21	E8-EYFP	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  22	E9-EYFP	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  23	E10-ECFP	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  24	F1-ECFP	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  25	F2-ECFP	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  26	F3-ECFP	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  27	F4-pBAD-TetR	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  28	F5-pBAD-TetR	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  29	G1-EYFP	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  30	G4-pSB-CEYFP	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  31	G6-pBAD(1)	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  32	G7-pBAD(2)	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  33	G8-N-term tag	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  33	G9-Lumazine	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  33	1kb ladder	0.5 ladder + 2 Dye (6X) + 9.5 Milli-Q H2O

  
  

  Results:

  June 28/2010

  (in lab: JV, HS, AV, HB)
  

  Objective:: To restrict, run 1% agarose gel, and ligate all mms6, lumazine, xylE into pET28(a) for overexpression tests.
  

  Method:
  1) Restrict all parts and pET28(a) with NotI
  

  2) Run on agarose gel (1%)
  

  3) Ligate parts into pET28(a)
  

  Parts:
  

  from working plasmid box 2010:
  

  • A3-Lumazine
  • B9-Mms6
  • D7-xylE
  • D8-xylE
  • I10-Mms6

  from pink tray:
  

  • B9-xylE
  • C4-xylE
  • G9-Lumazine

  (2X) pET28(a)
  

  Restrictions:
  

  • 2µL DNA
  • 2µL Orange Buffer
  • 0.25µL Enzyme (NotI)
  • 15.75µL Milli-Q H₂O

  Protocol:
  

  • 1) Incubate for 1 hour at 37^oC
  • 2) Heat kill for 20 minutes at 80^oC (heat block)
    

  Agarose Gel:
  

  Lane	Sample	Load (µL)
  1	1kb ladder	0.5 ladder + 2 Dye (6X) + 9.5 Milli-Q H2O
  2	Lumazine A3 RD	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  3	Lumazine A3	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  4	Mms6 I10 RD	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  5	Mms6 I10	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  6	Mms6 B9 RD	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  7	Mms6 B9	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  8	xylE D8 RD	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  9	xylE D8	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  10	xylE D7 RD	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  11	xylE D7	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  12	Lumazine G9 RD (p)	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  13	Lumazine G9 (p)	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  14	xylE B9 RD (p)	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  15	xylE B9 (p)	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  16	xylE C4 RD (p)	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  17	xylE C4 (p)	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  18	pET28(a) RD	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  19	pET28(a) RD	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O
  20	pET28(a)	5 DNA + 2 Dye (6X) + 5 Milli-Q H2O

  Ran gel at 100V for 90 minutes
  

  Results:pET28(a) bands are all smeared, and are not useful from inserting parts.
  
  

  June 30/2010

  (in lab: JV)
  

  Objective:: Isolate plasmid DNA from DH5α
  

  Method:Used Kothe Maxiprep protocol.
  Cell Pellet Weights:
  

  • xylE = 1.15g
  • Mms6 = 1.13g
  • pET28(a) = 0.67g
  • lumazine synthase = 1.1g
    

  Results:
  

  Retrieved from "http://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/June"