Team:WashU/Notebook/MolecularBiology

From 2010.igem.org

Latest revision as of 06:52, 27 October 2010

2010/07/01

• Plated The Megax BH10B strain and Strain 8 from the cohen lab onto an amp plate to check that the Megax BH10B strain is killed

2010/07/13

• Minipreped all the cultures and nanodropped them
• Ran 3 PCR reactions (@ 58,60, and 62 C) with primers p1 and p2 in order to attach the kozak onto the YFP
• Enzyme digested Nat (bglII and EcoRI), Kan (BamHI and EcoRI), and Promoter (EcoRI). Did 2 reactions each with ~500 ng DNA

2010/07/14

• Gel from July 13th

• Lanes from top to bottom:

1)   Ladder
2)   5uL of 58 degree YFP + 1uL of loading dye
3)   5uL of 60 degree YFP + 1uL of loading dye
4)   5uL of 62 degree YFP + 1uL of loading dye
5)   5uL of KanMx4 1 + 1uL of loading dye
6)   5uL of KanMx4 2+ 1uL of loading dye
7)   5uL of NatMx4 1 + 1uL of loading dye
8)   5uL of NatMx4 2+ 1uL of loading dye
9)   5uL of Promoter 1 + 1uL of loading dye
10) 5uL of Promoter 2 + 1uL of loading dye

2010/07/15

• Gel reads from left to right as follows:

Dna Ladder; 
NatMX4-Promoter construct 2x; 
KanMX4 plasmid backbone 2x.

• DNA ladder information is here at http://www.neb.com/nebecomm/products/productN3232.asp

2010/07/16

• Transformed EColi with the Kan plasmid containing the NatMX4 Promoter sequence.
• Bacteria Transformation July 16, 2010

1)	Thaw competent cells on ice right before transformation.
2)	Make 3 100uL tubes of competent cells
3)	Add 4 ul of plasmid to cells, flick gently.
4)	Incubate on ice 30 min.
5)	Heat-shock cells for 30 seconds at 42oC w/o shaking
6)	Transfer to ice, incubate 2 min
7)	add 250 ul room temp SOC Media
8)	Shake @ 37o for 1 hr
9)	Spread 150 ul on a pre-warmed ampicilin selector plates, allow sample to dry on plate
10)	Incubate o/n at 37oC upside down

2010/07/19

• Transformation of NatMX4/Promoter vector failed.
• Analysis of gel purification showed that the separate pieces which were ligated together on Thursday July 15th were of the correct length.
• Conclusion: the ligation failed; most likely due to denatured ligase. The ligase icebath had melted prematurely and heat inactivated the enzyme.
• Yeast group obtained several replacement ligases and necessary buffers from the Cohen Laboratory in the 4444 building of the WashU Med School.

• Transformation of C2 and SxL constructs into E. coli (AH)
  • Reconstituted 5ug of each construct (in plasmid) in 10uL dH20. Final conc. of this stock solution is 500ng/uL. 10uL of working stock of each solution was made by diluting stock solution 1:10. Final conc. of working stock is 50ng/uL.
  • Thaw 300uL tube of DH5alpha competent cells and divide among 5 tubes (60uL each). Amounts of 50ng/uL working stock were added to tubes labeled A-E in the following way:

A: 1uL C2 
B: 5uL C2
C: 1uL SxL
D: 5uL SxL
E: Control=no DNA

• • Incubate on ice 30 min, heat shock for 30s at 42C, rest on ice for 2 min.
  • Add 250uL of LB to each tube, Shake at 37C for 1hr
  • Plate 150uL on warm LB + Amp plates o/n at 37C

2010/07/20

• Digested 4 sequences:

1) NatMx4 with BglII and EcoRI: in microliters [5 buffer 3(NEB); 2.5 DNA at 200 ng/uL; 0.5 of BSA; 
   1 of each enzyme; 40 of H2O.]
2) Constitutive Promoter with EcoRI and SpeI: in uL [5 buffer 4(NEB); 2.5 DNA at 200 ng/uL; 0.5 of BSA; 
   1 of each enzyme; 40 of H2O.]
3) Construct 2 with BglII and XbaI: in uL [5 buffer 2(NEB); 1 DNA at 500 ng/uL; 0.5 of BSA; 1 of each enzyme; 
   41.5 of H2O.]
4) SxL with BamHI: in uL [5 buffer 3(NEB); 1 DNA at 500 ng/uL; 0.5 of BSA; 1 of BamHI; 41.5 of H2O.]

• all were incubated for 4 hours followed 20 minutes of heat inactivation.

2010/07/21

• Digest SxL construct with EcoRI: added 1uL EcoRI to already done mix from 20 July digestion.
• Gel purify NatMx4, Promoter, Construct 2, and KanMx4 digest results from 20 July 2010

      Note: KanMx4 bands were not observed but approximate region of gel was removed for ligation KSB.

• Ligate all three pieces together: NatMx4, Promoter, and Construct 2
  • 2 reactions:

       1) NCPA = NatMx4+Promoter+Construct 2
                       3.5uL Nat + 3.5uL Pro + 1.5uL C2
       2) NCPB = NatMx4&Promoter+Construct 2     
         [NatMx4&Promoter was pre-existing ligation product from 15/16 Jul]
                       7uL NP + 1.5uL C2

• Ligate KanMx4 with Sxl
  • 2 reactions:

       1) KSA = KanMx4 Digest 2 + SxL
                       7uL KanDigest2 + 1.5uL SxL
       2) KSB = KanMx4 (purified) + SxL
                       7uL GelPureKanMx4 + 1.5uL SxL

• Freezer stocks of C2 and SxL
• 1:1 ratio of LB was mixed with 100% glycerol to make a 50% glycerol/LB solution.
• 750uL of glycerol/LB solution was added to 750uL of each C2 and SxL culture from transformation on 2010/07/19.
• Two tubes of each were placed at -80C.

2010/07/22

• Transformed the ligation DNA from reactions NCPA, NCPB, KSA, and KSB into E. Coli using the following protocol.
• Bacteria Transformation

1.      Thaw competent cells on ice right before transformation.
2.      Add 4ul of plasmid to cells, flick gently.
3.      Incubate on ice 30 min.
4.      Heat-shock cells for 30 seconds at 42oC w/o shaking
5.      Transfer to ice, incubate 2 min
6.      add 125 ul room temp LB Media
7.      Shake @ 37o for 1 hr +
8.      Spread 100 ul on a pre-warmed plate w/ correct antibiotic selector, allow sample to dry on plate
9.      Incubate o/n at 37oC upside down

2010/07/26

• Digest and gel purify to confirm the results of the SxL construct transformants created on July 22.
• Gel image is attached:

• Reactions 4, 5, 6, and 7 of the Yeast Group's gel are from July 21 ligation, July 22 transformation, and the July 24 miniprep of the July 23 cultures. [The leftmost lane is a ladder; numbering begins at the second lane with Number 1.]

• Sequencing the YFP to confirm the Kozak Sequence:
  • iGEM1 YFP 62 degrees C primer P9
  • iGEM2 YFP 62 degrees C primer P2
  • iGEM3 YFP 62 degrees C primer P9
  • iGEM4 YFP 62 degrees C primer P2

2010/07/27

• Digestion of Terminator and YFP pieces (YFP is currently being sequenced to confirm Kozak Sequence)
• Terminator

•	37.5 uL of H20
•	5 uL of NEB buffer 3
•	5 uL of Terminator DNA
•	1 uL of Pst1 enzyme
•	1 uL of Xba1 enzyme
•	0.5 uL of BSA

• YFP

•	37.5 uL of H20
•	5 uL of NEB buffer 2
•	5 uL of YFP DNA
•	1 uL of Pst1 enzyme
•	1 uL of Spe1 enzyme
•	0.5 uL of BSA

• Gel Purification and Confirmation of NPCA, NPCB, Terminator, and YFP

• Gel Lanes:

1) Ladder
2) NPCA 1
3) NPCA 2
4) NPCB 1
5) NPCB 2
6) Terminator
7) YFP
8) YFP

2010/07/28

• Double Digests of KSA2 (confirmed); NPCA 1 & 2; and NPCB 1 & 2.

•	32.5 uL of H2O
•	10 uL of DNA
•	5 uL of NEB buffer 4
•	1 uL of XhoI
•	1 uL of AvrII
•	0.5 uL of BSA

• Began Colony PCR of NPCA and NPCB plates: 14 colonies taken from each.

2010/07/29

• Colony PCR:
• Primers p1 and p5 are defective Colony PCRs will not yield proper results
  • The back up cultures still are intact and can be reused when the proper primers arrive

• Digestions:

• • YFP Biobrick:

•	32.5 uL of H2O
•	5 uL of DNA
•	5 uL NEB buffer 3
•	5 ul CIP
•	1 uL PstI
•	1 uL XbaI
•	0.5 uL BSA

• • YFP kozak [Mix is defective due to Primer 1 error; DNA does not contain XbaI site]

•	30 uL kYFP DNA
•	12.5 uL of H2O
•	5 uL NEB buffer 4
•	1 uL XbaI
•	1 uL SpeI
•	0.5 uL BSA

• • C2 control

•	37.5 uL H2O
•	5 uL DNA
•	5 uL NEB buffer 4
•	1 uL XhoI
•	1 uL AvrII
•	0.5 uL BSA

• Gel:

•	Lane 1: Ladder 1kb
•	Lane 2: C2 control
•	Lane 3: NPCA1         3.7 and 2.3kb
•	Lane 4: NPCA2         3.7 and 2.3kb
•	Lane 5: NPCb1         3.7 and 2.3kb
•	Lane 6: NPCb2         3.7 and 2.3k

• • Conclusion NP was not present within the construct 2 plasmid

• Gel 2:

• • YFP backbone--- not observed Gel tossed out

• Ligations:

• • NPCc: ligate C2// BglII & XbaI with CIP A with NatMx4 and Promoter

•	1uL Y4 ligase buffer
•	0.5 uL ligase
•	3 uL NatMx4
•	3 uL Promoter 
•	2.5 uL C2

• • NPb: NatMX4 ligates to Promoter

•	17 uL NatMx4
•	17 uL Promoter
•	4 uL T4 ligase buffer
•	2 uL ligase

• Extra Digestion:

• • C2 // bglII & XbaI CIP B

•	27.5 uL H2O
•	10 uL DNA
•	5 uL CIP
•	5 uL NEB buffer 3
•	1 uL BglII
•	1 uL XbaI
•	0.5 uL BSA

2010/07/30

• Gel purify 30 uL NPb and 30 uL C2b
• Make NPCd: Ligate NPb & C2b (non purified) ~ 10 uL of each

•	5.5 uL NPb
•	3 uL C2b
•	0.5 ligase
•	1 uL buffer

• Make NPCe: Ligate NPb & C2b (purified)

•	24 uL of NPb DNA
•	1.5 uL of C2b DNA
•	3 uL buffer
•	1.5 uL ligase

• Re-attempt ligation of NatMx4 and Promoter

•	25.5 uL mix of NatMx4 and Promoter
•	3 uL buffer
•	1.5 uL ligase

2010/08/02

• Transformations: NPCc NPCd NPCe and KSA2

1.	Thaw competent E.Coli cells on ice right before transformation.
2.	Make 4 tubes with 70uL of competent cells each
3.	Add 3 ul of NPCc, d and e plasmid to 3 tubes and 1 uL of KSA2 to the last tube, flick gently.
4.	Incubate on ice 30 min.
5.	Heat-shock cells for 30 seconds at 42oC w/o shaking
6.	Transfer to ice, incubate 2 min
7.	add 140 ul room temp LB Media
8.	Shake @ 37o for 2 hr [to ensure recovery from shock]
9.	Spread 100 ul on a pre-warmed ampicilin selector plates, allow sample to dry on plate
10.	Incubate o/n at 37oC upside down

• PCR reactions:

• • Reaction 1st round

kYFPa: 94ng of YFP at 59 degrees C using p10 & p2   
kYFPb: 94ng of YFP at 61 degrees C using p10 & p2

• • Reaction 2nd Round

kYFPa1: 1 uL of kYFPa at 59 degrees C using p11 & p2 
kYFPb1: 1 uL of kYFPb at 59 degrees C using p11 & p2
kYFPa2: 1 uL of kYFPa at 61 degrees C using p11 & p2
kYFPb2: 1 uL of kYFPb at 61 degrees C using p11 & p2

2010/08/03

• Cultures:
  • Created liquid cultures from the transformation results of Ksa2, NPCc, NPCd, NPCe, and C2.

               [C2 transformation was performed by Amanda when the C2 construct arrived]

• Digests: kYFPa1, a2, b1, b2 and YFPBiobrick on backbone pSB1C3
  • For ALL:

•	27.5 uL of H2O
•	15 uL of DNA of the respective construct
•	5 uL of NEB buffer 3
•	1 uL of PstI enzyme
•	1 uL of XbaI enzyme
•	0.5 uL of BSA

• • [resulting products were column purified and eluted to 50 uL]

• Ligation of kYFP's and the backbone

•	21 uL of kYFPa1, a2, b1, b2 in 4 respective tubes
•	4.5 uL of BB into all four tubes
•	3 uL buffer 
•	1.5 uL T4 ligase

• Of NatMx4 and Promoter

• • NatMx4

•	35 uL of H2O
•	7.5 uL of DNA
•	5 uL of NEB buffer 3
•	1 uL of bglII
•	1 uL of EcoRI
•	0.5 uL BSA

• • Promoter

•	35 uL of H2O
•	7.5 uL of DNA
•	5 uL of NEB buffer 4 
•	1 uL of EcoRI
•	1 ul of SpeI
•	0.5 uL BSA

2010/08/04

• Gel purification of NatMX4 and Promoter
• 4 lanes Nat and 4 lanes Pro
• Successfully extracted the lower of 2 visible lines from all lanes [top line was vector; bottom was Nat and Pro inserts]
• Miniprep: Kan-SxL construct and Construct 2
• Transformation: kYFPa1, a2, b1, b2 on the Biobrick Backbone.

1.	Thaw competent cells on ice right before transformation.
2.	Make 4 75uL tubes of competent cells
3.	Add 4 ul of plasmid to cells, flick gently.
4.	Incubate on ice 30 min.
5.	Heat-shock cells for 30 seconds at 42oC w/o shaking
6.	Transfer to ice, incubate 2 min
7.	add 150 ul room temp LB Media
8.	Shake @ 37o for 1 hr
9.	Spread 100 ul on a pre-warmed ampicilin selector plates, allow sample to dry on plate
10.	Incubate o/n at 37oC upside down

• Ligation: NatMx4 + Promoter

•	30 uL of DNA (mix of both parts; they were extracted together)
•	4 uL of Buffer
•	4 uL of H2O
•	2 uL of T4 ligase

2010/08/06

• Transformation: kYFP on the Biobrick Backbone.

1.	Thaw competent cells on ice right before transformation.
2.	Make 1 100 uL tubes of competent cells
3.	Add 4 ul of plasmid to cells, flick gently.
4.	Incubate on ice 30 min. (10:45 to 11:15)
5.	Heat-shock cells for 30 seconds at 42oC w/o shaking
6.	Transfer to ice, incubate 2 min
7.	add  ul room temp SOC Media
8.	Shake @ 37o for 1 hr 
9.	Spread 100 ul on a pre-warmed ampicilin selector plates, allow sample to dry on plate
10.	Incubate o/n at 37oC upside down

2010/08/09

• Make Tetracycline plates to select for kYFP transformants
• Transform kYFP into E.Coli

1.	Thaw competent cells on ice right before transformation.
2.	Make a 75 uL tube of competent cells
3.	Add 2 ul of plasmid to cells, flick gently.
4.	Incubate on ice 30 min. (12:10 to 12:40)
5.	Heat-shock cells for 30 seconds at 42oC w/o shaking
6.	Transfer to ice, incubate 2 min
7.	add 125 ul room temp SOC Media
8.	Shake @ 37o for 1 hr
9.	Spread 100 ul on a pre-warmed tetracycline selector plates, allow sample to dry on plate
10.	Incubate o/n at 37oC upside down

2010/08/10

• Made a culture from possible colonies on tetracycline plate
• Double Digest of pSB1A3

•	H2O
•	DNA
•	NEB buffer 3
•	XbaI
•	PstI
•	BSA

• Gel purification of digest products
  • 1kb ladder, 3 lanes of pSB1A3, and 3 lanes of YFP-BB
  • results: all 3 bands observed (one for the pSB1A3 digest and 2 for the YFPbb) [an extra band from the biobrick was seen above the 2 desired YFP bands possibly single or uncut plasmid]
• Gel extraction of digest products
  • 1 extraction for each of the 3 desired bands

• Ligations

•	kYFP to pSB1A3
•	kYFP to Biobrick backbone
•	YFP to pSB1A3
•	YFP to Biobrick backbone

• Transformation of the ligation products into E. Coli

1.	Thaw competent cells on ice right before transformation.
2.	Make 4, 70 uL tube of competent cells
3.	Add 2 l of plasmid to cells, flick gently.
4.	Incubate on ice 30 min. (3:45 to 4:15)
5.	Heat-shock cells for 30 seconds at 42oC w/o shaking
6.	Transfer to ice, incubate 2 min
7.	add 125 l room temp SOC Media
8.	Shake @ 37o for 1 hr
9.	Spread 100 l on a pre-warmed ampicilin selector plates, allow sample to dry on plate
10.	Incubate o/n at 37oC upside down

2010/08/11

• Colony PCR

•	4 possible transformant colonies PCR'd and back-up culture made

• PCR

•	Re-PCRing the kYFP at 59, 60, 62 degrees C [product was named kYFP2]

• Transformation of kYFP with o/n ligation results

•	50uL of cells
•	2 uL of kYFP on Biobrick backbone
•	2 uL of kYFP on pSB1A3
•	150uL of SOC media
•	plated all of the incubated/shaken culture

• Gel purifications
  • 100b ladder; 4 lanes of colony PCR; 59*C PCR of new kYFP; 60*C PCR of new kYFP; 62*C PCR of new kYFP.
  • results:

• Digests of PCR Products and the Terminator
• Digesting PCR kYFP with XbaI and SpeI
• Digesting terminator [in vector] with XbaI and CIP
• Ligation

2010/08/12

• Miniprepped the cultures made on the 11th [#4 had no growth or pellet]
• Created cultures of the 4 transformant colonies from the 11th's transformation
• Redesigned the kYFP reverse primer
• Ligate Terminator and kYFP2 (from the 11th's digestion)
• [30min room temp ligation]
• PCR at 60*C the Miniprep results, a YFP control and the Ligation product kYFP2term with primers p2 and p12& p9 and p12 reactions with the YFP control and the kYPF2term. [7 rxns total as #4 yielded no pellet for miniprepping]
• [p2 was the questionable reverse kYFP primer]

• Gel run: 100bp ladder, culture 1 DNA; cult. 2 DNA; cult. 3 DNA; YFP 2-12 PCR; YFP 9-12 PCR; kYFP2 2-12 PCR; kYFP2 9-12 PCR
  • Results

• • Conclusion: Colonies were not desired kYFP transformants [match the pattern of the YFP (2-12) control]

• Transformation of E.Coli with the kYFP2term plasmid (ligation product)

   3uL in 50uL of cells
   150uL SOC media
   75 ul plated onto amp selector plates

2010/08/13

• Transformation yielded a large number of colonies which may contain the kYFP2term construct
• Pour new ampicilin plates
• Beginning screening processes today:
• Grow 16 cultures of several colonies from transformants
• Future screening steps:
  • Miniprep the cultures
  • Digest DNA with XbaI & PstI
  • Digest C2 with XbaI & PstI
  • Gel12 purify C2 and the correct kYFP2term from the improper kYFP2term [X will cut improper into 3 pieces, correct into 2]
  • 30min room temp ligate correct kYFP2term with C2
  • Transform the ligated construct, C2kYFP2term, into E. Coli
  • Grow cultures from transformation
  • Miniprep the cultures
  • Submit DNA for sequencing
  • Digest with bglII & XbaI & CIP
  • Gel purify
  • 30 min room temp ligation of C2kYFP2term with the NatMX4-Promoter construct
  • Transform ligation product into E.Coli

2010/08/14

• Miniprepped the cultures (16) made from kYFP2term transformants
• Digests:
  • Digested C2 with SpeI and PstI and CIP in NEBuffer 2. [27.5 H2O; 14 DNA 1 CIP; 1 SpeI; 1 PstI; 5 buffer; 0.5 BSA]
  • 6 Digests of Miniprepped DNA with XbaI and PstI in NEBuffer 2 [35.5 H2O; 7 DNA; 5 buffer; 1 XbaI; 1 PstI, 0.5 BSA]

               Note: rxns 12,13,14,15, and 16 used low concentration XbaI

• Gels:
  • 12 lane gel contained: 1kb ladder; culture samples 1-11
  • 8 lane gel contained: 1 kb ladder; culture samples 12-16
• Expected results

1. Successful construction: 2 kb band and .993kb band
2. Backwards kYFP2: 3 kb band
3. No kYFP2: 2kb band and .25 kb band

• Results

• Conclusion: #3 results for all samples; no kYFP2 ligated into the terminator

2010/08/19

• Got the new primers in today.
• Ran a PCR to make the new KYFP inserts.
• Used 1 ul of YFP biobrick plasmid as the template.
• p15 and p 16 were used to make the KYFP3.
• p10 and p17 were used to make the preKYFP4.
• Three PCR reactions were set up for each and were then run at 58, 60, and 64 C with 1 min extension time.
• The PCR products were then run out on a gel.
• The bands coresponding to ~ 800 bp were cut out and gel purified.

• Lanes:

1        100 bp ladder
2,3      KYFP3 @58
4,5      KYFP3 @60
6,7      KYFP3 @64

• Lanes:

1        100 bp ladder
2,3      preKYFP4 @58
4,5      preKYFP4 @60
6,7      preKYFP4 @64
8        100 bp ladder

• The preKYFP4 products were cleaner as was to be expected.
• The KYFP3 @60 has anomalous lower bands ~200 bp and the KYFP3's in general have more minor banding patterns

• Running a PCR over night.

•	1 ul of KFYP3 64+p13+p16=KYFP5
o	running 2 reactions: one at 60 and the other at 62 C
•	1 ul of preKYFP4 64+p11+p16=KYFP4
o	running 2 reactions: one at 60 and the other at 62 C

2010/08/20

• Ran the PCR products out on a gel.

• Lanes:

1        100 bp ladder
2        KYFP5 @60
3,4     KYFP4 @60
5,6     KYFP5 @62
7,8     KYFP4 @62

• The bands that are ~800 bp were cut out and purified.
• Then I set up a five hour enzyme digest @ 37 C.
• I cut KYFP3 @64 w/ XbaI and PstI, KYFP4 @62 w/ XbaI and PstI, KYFP5 @62 w/ XbaI and PstI, YFP backbone w/ XbaI, PstI, and CIP, and Terminator with XbaI and CIP.
• The digested results were run out on a gel and then the appropriate bands were excised and the DNA was purified out.
• An over night RT ligation was set up with 1 ul of backbone: 7.5 ul insert.
• Cut KYFP3, KYFP4, and KYFP5 were ligated into the cut YFP back bone.
• A negative control (only cut YFP back) and a positive control (YFP insert back into YFP back bone) were also set up.

2010/08/26

• Results from transformation on 8/25

Number	Plasmid 	Plate 	Observations
1	pSB1AT3 	Tet 	 Lawn of colonies with sprinkled red transformants
2	None 		Tet 	 Lawn of colonies
3	pSB1AT3 	Amp 	 ~500 colonies, all small, mostly red transformations but some
4	None 		Amp 	 ~100 colonies, some very large in size
5	pSB1K3 		Kan 	 ~80 colonies observed all red transformants
6	None 		Kan 	 No colonies observed