User:Perry/Spring 2007

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2/12/07

dissolved 500mg ampicillin salt into 10ml water to prepare 50mg/ml ampicillin stock; aliquotted 1ml into tubes, left in -20dC.

overnight 1ml LB-amp cultures of JLO159, W, H, S, ~4:30pm.

2/13/07

took overnight cultures out ~7:30pm. diluted 1:10, measured OD600.

159- .335, W- .245, H- .231, S- .279

made liquid cultures of 5ml LB-amp + 250ul overnight cultures. incubated 3h, added 5mM IPTG (53ul 0.5M), incubated 2h, diluted 1:10, measured OD600.

159- .106, W- .044, H- .023, S- .057

Aliquotted 1.5mL of H into each tube and proportional volumes of the rest. aliquotted volumes into tubes. centrifuged 8000rpm, 3min. aspirated. reconstituted in 10ul streptavidin aptamer, 10ul B/F hybrid, or 10ul B0/F hybrid.

4uM streptavidin aptamer: 48ul PBS + 2ul streptavidin aptamer (100uM)

4uM B/F hybrid: 46ul PBS + 2ul Bside oligo (100uM) + 2ul Iside oligo (100uM)

4uM B0/F hybrid: 46ul PBS + 2ul DispI oligo (100uM0 + 2ul Iside oligo (100uM)

Wrapped tubes in a napkin, rotated on rotisserie at room temperature for 1h. washed in PBS 3X, reconstituted in 50ul PBS, loaded into clear 96-well plate.

2/28/07

I'm going to shift the project away from streptavidin for now, until I learn more about membrane fractionation or until I get a full streptavidin clone from Takeshi Sano. I'm going towards expressing PDZ repeats on the surface.

Prepared 150ml cultures of JLO66 and JLO159.

3/1/07

Hi-Speed Midiprep of JLO66 and JLO159.

3/2/07

Received primers PDZ1F, PDZ2F, PDZ1RS, PDZ2RS. Dissolved in (10x#nmol)ul for 100uM. Diluted 1:10 for 10uM used in PCR.

Received F11/Na template from Alain; nanodropped at ~10ng/ul. Made a 1:50 dilution.

Prepared six 50ul PCR reactions, adding the 1ul of each of the following to 47ul Platinum PCR Supermix.

PDZ1: F11, PDZ1F, PDZ1RS

PDZ2: F11, PDZ2F, PDZ2RS

PDZ1+2: F11, PDZ1F, PDZ2RS

PDZ1/2/1+2 (1:50): same, using 1:50 F11.

Incubated 95dC 10min, (95dC 30sec, 55dC 30sec, 72dC 60sec)x30, 72dC 10min.

Walker ran 10ul of each PCR reaction on an e-gel.

Lane 1: 1kb+

Lane 5: PDZ1

Lane 6: PDZ2

Lane 7: PDZ1+2

Lane 8: PDZ1 (1:50)

Lane : PDZ2 (1:50)

Lane 10: PDZ1+2 (1:50)

3/5/07

Repeated PDZ1 and PDZ1+2 PCRs (50ul) with F11, DC1-2/Na (~200ng/ul), and DC1-2/Na(1:20). Changed annealing temperature to 45dC. Ran 10ul in an e-gel.

Lane 1: 1kb plus

Lanes 2, 3: F11 PDZ1 and PDZ1+2

Lanes 4, 5: DC1-2 PDZ1 and PDZ1+2

Lanes 6, 7: DZ1-2 (1:20) PDZ1 and PDZ1+2

Performed PCR purification with yesterday's F11 PDZ2 and today's F11 PDZ1 and PDZ1+2. Eluted in 30ul water.

Performed Topo cloning/transformations with PCR-purified PDZ1, 2, 1+2.

3/6/07

Made streaks and M13F/R colony PCRs of PDZ1 (1, 2, 3, 4), PDZ2 (1, 2, 3, 4), and PDZ1+2 (2, 3, 4).

Lane 1: 1kb+

Lanes 2-5: PDZ1 colony PCR #1, 2, 3, 4

Lanes 6-9: PDZ2 colony PCR #1, 2, 3, 4

Lanes 10-12: PDZ1+2 colony PCR #2, 3, 4

Prepared two 3ml cultures of PDZ1 #1, PDZ2#1, PDZ1+2 #3.

3/7/07

Performed minipreps, sent out sequencing, prepared XbaI/PstI digests of PDZ1, 2, 1+2. also, SpeI/PstI digests of JLO66 and JLO159.

3/8/07

Ran 1% 100ml agarose gel (used TAE, and 3ul ethidium bromide), 140V, 50 minutes.

Lane 1: 1kb+

(Lane 2: undigested LppOmpA/pET29b)

Lanes 3, 4: PDZ1 X/P digest

Lanes 5, 6: PDZ2 X/P digest

Lanes 7, 8: PDZ1+2 X/P digest

Lanes 9, 10: JLO66 S/P digest

Lanes 11, 12: JLO159 S/P digest

(Lanes 13, 14: Lpp-OmpA/pET29b NheI/PstI digest)

Cut out 300bp band from Lanes 3 and 5, 500bp band from lane 7, and 4kb bands from Lanes 9 and 11. Performed Qiaquick gel extraction, added the sodium acetate step, eluted in 50dC water, 30ul. nanodropped then and got all 15-20 ng/ul, except for JLO159, ~35ng/ul.

Cut out same bands from Lanes 4, 6, 8, 10, 12, performed Montage gel extraction, and got all 2-3ng/ul.

Performed Quick Ligation with 4ul vector and 8ul insert, plus 12ul ligase buffer and 1ul quick ligase. Transformed into 18ul Top10 cells.

  • JLO66 and PDZ1
  • JLO66 and PDZ2
  • JLO66 and PDZ1+2
  • JLO159 and PDZ1
  • JLO159 and PDZ2
  • JLO159 adn PDZ1+2

3/9/07

Ug, none of the ligations worked. Onto the Clonewell!

3/11/07

Prepared 150ml LBamp cultures of PDZ1, 2, 1+2. Incubated 200rpm, 37dC, 8pm.

3/12/07

Performed midipreps. Set up 50ul, 2ug XbaI/PstI digests of PDZ1, 2, 1+2, and SpeI/PstI of JLO66, JLO159.

3/13/07

Ran digests on Clonewell e-gel, split each digest into 2 wells, 25ul each.

Nanodropping collections all yielded ~20ng/ul; however, nanodropping a collection from a blank lane yielded ~16ng/ul. Running 20ul of JLO66, JLO159 collections on a 1.2% e-gel yielded strong, sharp bands. Tried out ligation/transformations 2.5:7.5 vector:insert.

Set up new digests, same as yesterday.

3/14/07

Ligation/transformations failed. Retried Clonewell e-gel. Vacufuged samples to up concentration to about 50ng/ul.

3/15/07

Got colonies! Variation in colony sizes, possibly due to the fact that I transformed in Top10 cells, not Top10F'.

Prepared VF2/VR (200nM) PCRs and plate streaks from two big colonies (#1 and 2) and two smaller colonies (#3 and 4). Incubated 1m-1m-1m30s.

3/16/07

Ran 1.2% egels of all PCRs.

Lane 1: 1kb+

Lanes 2-5: JLO66PDZ1 PCRs #1-4

Lanes 6-9: JLO159PDZ1 PCRs #1-4

Lane 1: 1kb+

Lanes 2-5: JLO66PDZ2 PCRs #1-4

Lanes 6-9: JLO159PDZ2 PCRs #1-4

Lane 1: 1kb+

Lanes 2-5: JLO66PDZ1+2 PCRs #1-4

Lanes 6-9: JLO159PDZ1+2 PCRs #1-4

Parts sizes are as follows...

  • J04500 - 220
  • Lpp29 - 87
  • OmpA66 - 63
  • OmpA159 - 339
  • PDZ1 - 261
  • PDZ2 - 261
  • PDZ1+2 - 546

Construct sizes are as follows (sum of parts plus 6bp in between parts)...

  • JLO66PDZ1 - 649
  • JLO66PDZ2 - 649
  • JLO66PDZ1+2 - 934
  • JLO159PDZ1 - 925
  • JLO159PDZ2 - 925
  • JLO159PDZ1+2 - 1210

VF2 primer anneals ~140bp upstream, and VR primer anneals ~100bp downstream of the cloning site.

3/23/07

After I retried digests, this time with ~3ug in 2x25ul digests, and clonewelled, I redid ligation-transformations.

3/30/07

Alain inoculated cultures from colonies, performed minipreps, diluted 1:100, and performed VF2-VR PCRs.

Lanes 1 to 6: PCR, JLO159-PDZ1 #1-6

Lane 7: 1kb+

Lanes 8 to 10: JLO66-PDZ1 #1-3

Lanes 1 to 3: JLO66-PDZ1 #4-6

Lane 4: 1kb+

Lanes 5 to 10: JLO159-PDZ1+2 #1-6

Approximate PCR sizes are as follows (sum of parts plus 6bp in between parts plus 240bp VF2/VR)...

  • JLO66PDZ1 - 889
  • JLO66PDZ2 - 889
  • JLO66PDZ1+2 - 1174
  • JLO159PDZ1 - 1165
  • JLO159PDZ2 - 1165
  • JLO159PDZ1+2 - 1450

4/10/07

Wow, okay, back in lab. Let's look at Alain's sequences.

  • JLO66PDZ1 #6 is correct, beginning to end.
  • JLO159PDZ1 #6 is correct from middle of PDZ1 domain to end. The PDZ1SR reverse sequencing reaction failed after <200bp.
  • JLO159-PDZ1+2 #1 is correct from middle of JLO159 to near end of PDZ1+2.
  • JLO159-PDZ1+2 #3 is correct from middle of JLO159 (a little farther upstream than #1) to near end of PDZ1+2. The orientations of the sequence alignments was weird.

Sent out the following sequencing reactions.

  • 113: JLO159PDZ1 #6 OmpA46F
  • 114: ...VR
  • 115: JLO159PDZ2 #3 PDZ2F
  • 116: ...PDZ2RS
  • 117: JLO159PDZ2 #6 PDZ2F
  • 118: ...PDZ2RS
  • 119: JLO66PDZ2 #1 PDZ2F
  • 120: ...PDZ2RS
  • 121: JLO66PDZ2 #5 PDZ2F
  • 122: ...PDZ2RS
  • 123: JLO66PDZ1+2 #5 PDZ1F
  • 124: ...PDZ2RS

4/16/07

Checked the sequencing results from 4/10/07, and they all check out. So along with 3/23 sequencing, the following have been confirmed.

JLO66PDZ1 #6 JLO66PDZ2 #1 or #5 JLO66PDZ1+2 #5 JLO159PDZ1 #6 JLO159PDZ2 #6 JLO159PDZ1+2 #1

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