User:Perry/Summer 2007

6/25/07

Received hisGFP_F_BB and GFP_R_BB primers, reconstituted at 100uM and diluted to 10uM, prepared PCRs with 45ul Supermix, 2ul hisGFP_F_BB (10uM), 2ul GFP_R_BB (10uM), 1ul pEGFP (1:1000 of 500ng/ul). Incubated 95dC 10min, (95dC 30s, 55dC 30s, 72dC 60s)x30, 72dC 10min, 4dC hold.

Previously performed 25ul digests using 21.25ul miniprep, 0.25ul BSA(100x), 0.5ul enzyme 1, 0.5ul enzyme 2, 2.5ul Buffer 2. Incubated 37dC 12h, 80dC 20min, 4dC hold.

• R0052 XbaI/PstI
• J04500 SpeI/PstI
• J04450 XbaI/PstI
• R0011-J06702 XbaI/PstI
• PDZ1 in Topo, XbaI/PstI
• PDZ2 in Topo, XbaI/PstI
• PDZ1+2 in Topo XbaI/PstI

Tried Clonewell. Gave up.

No bands for GFPmek PCR. Prepared 2 more PCRs with 10 cycles annealing at 45dC, and 20 cycles annealing at 55dC.

Reconstituted <bbpart>BBa_I3263</bbpart>, <bbpart>BBa_I3273</bbpart>, <bbpart>BBa_F1610</bbpart> in 15ul water, and transformed 1ul in 20ul Top10 cells. We will try to use these in a quorum sensing project.

6/26/07

Previously induced JLO66, -PDZ1, 2, 1+2, JLO159, -PDZ1, 2, 1+2...overnight, 1:40 dilution, 3h growth, add 1mM IPTG, 2h growth. Took out 100ul, spin down, resuspend in 100ul PBS.

Ran SDS-PAGE gel, 4ul of resuspended cells, 16ul water, 5ul sample buffer (5X). 150V for about 90min. Transferred to nitrocellulose with semi-dry blotting (20V for 30min). Stained with Ponceau, and saw very, very light bands. Left in blocking buffer overnight, rocker, 4dC.

Lane 1: SeeBlue2 ladder

Lanes 2-5: JLO66, -PDZ1, PDZ2, PDZ1+2 induced

Lanes 6-9: JLO159, -PDZ1, PDZ2, PDZ1+2 induced

Lanes 10-12: JLO159-PDZ1, PDZ2, PDZ1+2 not induced

Previously prepared 4.5ml liquid cultures of R0052, J04500, J04450, R0011-J06702, PDZ1, PDZ2, PDZ1+2. Did minipreps.

Prepared 25ul digests with 20.25ul DNA, 2.5ul Buffer 2, 1ul Enzyme 1, 1ul Enzyme 2, 0.25ul BSA.

• R0052 SpeI/PstI
• R0052 XbaI/PstI
• J04500 SpeI/PstI
• J04450 XbaI/PstI
• R0011-J06702 XbaI/PstI
• PDZ1 in Topo, XbaI/PstI
• PDZ2 in Topo, XbaI/PstI
• PDZ1+2 in Topo XbaI/PstI

Added 3ul Antarctic Phosphatase Buffer and 2ul Antarctic Phosphatase to R0052 SpeI/PstI, R0052 XbaI/PstI, J04500 SpeI/PstI.

Ran 1% 100ml agarose gel, 130V for 45min.

Lane 1: 1kb+

Lane 2-3: GFPmek PCR

Lane 4-6: PDZ1, 2, 1+2 XbaI/PstI

Lane 7: J04450 XbaI/PstI

Lane 8: R0011-J06702 XbaI/PstI

Lane 9: R0052 XbaI/PstI

Lane 10: R0052 SpeI/PstI

Lane 11: J04500 SpeI/PstI

Smears probably result from huge amounts of DNA in the digest. Cut out ~300bp insert bands from PDZ1 and PDZ2, 600bp insert band from PDZ1+2, ~1kb insert bands from J04450 and R0011-J06702, large vector bands from R0052 SpeI/PstI and XbaI/PstI, J04500 SpeI/PstI.

I NEED TO MAKE THIS GFPmek PCR WORK!!!

6/27/07

Incubated membrane with 5ml TBST (TBS + 0.1% Tween) + 5ml blocking buffer + 20ul anti-PDZ serum (1:500), 2h on shaker at room temp. Then washed 3x15min with TBST. Then incubated membrane with anti-mouse, anti-rabbit mix (1:15000) from Alain, 45min on shaker at room temp. Washed 2x10min TBST, 2x10min TBS, rinse with distilled water. Result? Crap. I didn't see any strong bands, maybe two very weak bands if I look hard enough.

Performed High Speed midiprep of BioBricks I13263, I13273, F1610. Sent off for sequencing with VF2 and VR primers.

A couple of days ago, I did a ligation of R0011-J06702 and J04450 (XbaI/PstI) into pSB2K3 vector (R0052 XbaI/PstI). I got a bunch of colonies that were red. Today, I did 5 streak/colony PCRs of each.

6/28/07

Ran colony PCRs on e-gel.

Lane 1: 1kb+

Lanes 2-6: R0011-J06702 streaks #1-5

Lanes 7-11: J04450 streak #1-5

George did a XbaI/PstI digest of the F1610, but the band did not appear, or there was a very faint one. Also, the sequencing came back, and the VF2 through VR sequence was about 300bp, while F1610 should be 800bp long. The first half of the sequence corresponded to the B0015 terminator at the end of F1610. George prepared another digest to incubate overnight.

I rewashed the membrane for several hours in TBST, and another hour in TBS, and Alain accompanied me to go scan it.

There seems to be a band in 6th lane and 9th lane from the ladder, which should be JLO159-PDZ1 induced and not induced, respectively.

Redid SDS-PAGE using 10ul of cells, 10ul water, 5ul sample buffer (5x), and this time with a positive control.

Lane 1: SeeBlue2 ladder

Lanes 2-5: JLO66, -PDZ1, PDZ2, PDZ1+2 induced

Lanes 6-9: JLO159, -PDZ1, PDZ2, PDZ1+2 induced

Lanes 10-11: JLO159-PDZ1, PDZ1+2 not induced

Lane 12: GST fusion protein from DC-1

Redid GFPmek PCRs, 7ul Supermix + 1ul template + 1ul forward primer (2uM) + 1ul reverse primer (2uM), using new 1:100 and 1:1000 dilutions from pEGFP stock, pairwise combinations between GFP_F/hisGFPmek_F_BB and GFPmek_R/GFKmek_R_BB, and a program with 15 cycles annealing at 45dC, 15 at 50dC, 16 at 55dC. Ran on a 1.2% e-gel.

Lane 1: 1kb+

Lane 2, 3: 1:100, 1:1000 pEGFP + GFP_F + GFPmek_R

Lane 4, 5: 1:100, 1:1000 pEGFP + hisGFPmek_F_BB + GFPmek_R_BB

Lane 6, 7: 1:100, 1:1000 pEGFP + GFP_F + GFPmek_R_BB

Lane 8, 9: 1:100, 1:1000 pEGFP + hisGFPmek_F_BB + GFKmek_R

GFP_F and GFKmek_R were the primers I used to PCR out GFP and add only the Mek2 sequence and nothing else. hisGFPmek_F_BB and GFPmek_R_BB primers were supposed to PCR out GFP and add a 5' His tag, a 3' Mek2 sequence, and flanking BioBricks sites.

Surprisingly (if i did everything correctly) this means that the problem isn't in hisGFPmek_F_BB but in GFPmek_R_BB. This also might mean that maybe I can do a PCR with hisGFPmek_F_BB/GFPmek_R that adds the His and prefix Biobrick sites and Mek2 sequence, but NOT the suffix Biobrick sequences: a BBhisGFPmek product. Then using this PCR product as a template, I can do a PCR with hisGFPmek_F_BB/GFPmek_R_BB, which should anneal better because the Mek2 sequence is already present in this new template.

6/29/07

Incubated membrane with 5ml TBST (TBS + 0.1% Tween) + 5ml blocking buffer + 20ul anti-PDZ serum (1:500, #743), 2h on shaker at room temp. Then washed 3x15min with TBST. Then incubated membrane with anti-mouse, anti-rabbit mix (1:15000) from Alain, 45min on shaker at room temp. Washed 2x10min TBST, 2x10min TBS, rinse with distilled water.

PCR purified the product from hisGFPmek_F_BB/GFPmek_R PCR. Took 10ul and ran on agarose gel. Made 50ul PCRs: 47ul Supermix, 1ul each of hisGFPmek_F_BB/GFPmek_R_BB primers (2uM), 1ul purified PCR product or 1ul 1:10 or 1ul 1:100 or 1ul 1:1000. Incubated 95dC 10min, (95dC 30s, 60dC 30s, 72dC 60s)x30, 72dC 10min, 4dC hold. PCR purified all 4 samples, and ran 10ul on an e-gel.

No bands, in either the first PCR or the second. Maybe I switched something in my test PCRs, or maybe things change when I up to 50ul. I redid the 10ul PCRs, two sets of them, with GFP_F/GFPmek_R_BB and hisGFPmek_F_BB/GFPmek_R. E-gel showed no bands.

7/2/07

Made the following 25ul digests, aimed for 1.2ug DNA in each.

• R0011 S/P
• R0040 S/P
• R0051 S/P
• S03608 E/S
• S03623 E/S
• J37034 X/P (x2)
• B0015 E/X

Added 3ul AP buffer, 2ul AP to R0011, R0040, R0051, B0015.

Ran 1% agarose gel, 130V, 45min.

Lane 1: 1kb+

Lanes 2-4: R0011, R0051, R0040 S/P

Lanes 5, 6: J37034 X/P

Lanes 8, 9: S03608, S03623 E/S

Lane 10: B0015 E/X

Cut out vector and insert bands, gel-extracted with Qiaquick. J37034 does not have a band at the right size, so Stephanie prepared a VF2/VR PCR and another XbaI/PstI digest, whic hwas incubated at 37dC overnight.

Prepared 20ul GFPmek PCRs, one test set, one set for template production. Used 1:100 of pEGFP plasmid or 1:100 E0240 miniprep (~100ng/ul). Incubated with 20 cycles annealing at 45dC, 20 at 50, 20 at 55, 20 at 60.

• GFP_F, GFPmek_R, pEGFP
• GFP_F, GFPmek_R_BB, pEGFP
• hisGFP_F_BB, GFPmek_R, pEGFP
• hisGFP_F_BB, GFPmek_R_BB, pEGFP
• bGFP_F, bGFPmek_R, E0240

Prepared 7ml LB-amp cultures of JLO159, -PDZ1, 2, 1+2 for miniprep and induction.

Sent off J23039 and T9002 for sequencing.

• AV01 J23039 VF2
• AV02 J23039 VR
• AV03 J23039 VF2
• AV04 J23039 VR

7/3/07

Made 1:40 dilutions for 1ml cultures, grew for 3h, induced a set and grew for 2h. Then OD'd and took out 400ul of the lowest OD (JLO159-PDZ1+2, induced), and adjusted volumes of the rest. Centrifuged, then resuspended in 100ul water. (JLO159-PDZ2, induced looked like I aspirated some of the cells, so I resuspended in 50ul). Left in freezer.

Also miniprepped the cultures, and then diluted to 1ng/ul and transformed 1ul into Top10 cells (for cells that don't need to be induced?)

Ran 1% agarose gel, 130V, 45min.

Lane 1: 1kb+

Lane 2: J37034 VF2/VR PCR

Lane 3: J37034 X/P digest

Lane 4: GFP_F, GFPmek_R, pEGFP PCR

Lane 5: GFP_F, GFPmek_R_BB, pEGFP

Lane 6: hisGFP_F_BB, GFPmek_R, pEGFP

Lane 7: hisGFP_F_BB, GFPmek_R_BB, pEGFP

Lane 8: bGFP_F, bGFPmek_R, E0240

J37034 is sooo wrong. I guess we'll have to reconstruct it from S03608/S03623 and E0240.

It looks something happened in Lanes 4, 6, 8. Let's try using 4 and 6 as templates in new PCRs. Made 50ul PCRs: 47ul supermix, 1ul each of hisGFP_F_BB and GFPmek_R_BB primer (10uM), 1ul of 4 or 6. Incubated one pair at 55dC annealing, one pair at 60dC annealing.

Performed ligation with Quick Ligase, 7ul S03608/S03623 + 3ul B0015 in pSB1AK3. Transformed into 20ul Top10.

7/5/07

Did two VF2/VR colony PCR from each JLO159-PDZ1, 2, 1+2 transformation in Top10 cells. Ran on e-gel, along with 10ul of the hisGFP_F_BB/GFPmek_R_BB PCRs.

Lane 1: 1kb+

Lanes 2, 3: JLO159PDZ1 colony PCRs, A and B

Lanes 4, 5: JLO159PDZ2 colony PCRs, A and B

Lanes 6, 7: JLO159PDZ1+2 colony PCRs, A and B

Lane 8: PCR with GFP_F/GFPmek_R product as template, 55dC annealing

Lane 9: PCR with hisGFP_F_BB/GFPmek_R product as template, 55dC

Lane 10: PCR with GFP_F/GFPmek_R product as template, 60dC annealing

Lane 11: PCR with hisGFP_F_BB/GFPmek_R product as template, 60dC

Looks like lanes 2, 5, 7, 8, 10 have right sizes.

From the remaining 40ul of PCR corresponding to lane 10, I took out 1ul and used it in a PCR, with 47ul supermix, 1ul hisGFP_F_BB, 1ul GFPmek_R_BB. I made two PCRs, one with 55dC annealing, and one with 60dC annealing, b/c I actually didn't prelabel the 55 and 60 yesterday, so I got them mixed up and am not sure if those designations are correct, so I don't know which annealing temperature produced a better band.

Ran induced cultures frozen from yesterday in SDS-PAGE. 10ul resuspended culture + 10ul water + 5ul sample buffer (5x). Also took a chip from DC-1 frozen pellet, resuspended in 100ul water, and used 2ul of it.

Lane 1: SeeBlue2 ladder

Lane 2: DC-1

Lanes 3-6: JLO159, -PDZ1, 2, 1+2, not induced

Lanes 7-10: JLO159, -PDZ1, 2, 1+2, induced

Transferred to membrane and left in blocking buffer, on rocker at 4dC.

Lane 2: DC-1

7/6/07

Yesterday I did some streaks and VF2/VR colony PCRs from the S03608-B0015 and S03623-B0015 ligation/transformations. Ran them on an e-gel today.

Lane 1: 1kb+

Lanes 2-6: S03608-B0015 colony PCRs #1-5

Lanes 7-11: S03623-B0015 colony PCRs #1-5

Also did the same with F2620 transformation. Tacked on the two GFPmek PCRs from yesterday.

Lane 1: 1kb+

Lanes 2-6: F2620 colony PCRs #1-5

Lane 7, 8: GFPmek PCRs with 55dC, 60dC annealing

Ug, this PCR is never going to work.

I redid the hisGFP_F_BB/GFPmek_R_BB PCRs using 4 and 6 templates from 7/3/07, annealing at 55dC or 60dC.

Lane 8: PCR with GFP_F/GFPmek_R product as template, 55dC annealing

Lane 9: PCR with hisGFP_F_BB/GFPmek_R product as template, 55dC

Lane 10: PCR with GFP_F/GFPmek_R product as template, 60dC annealing

Lane 11: PCR with hisGFP_F_BB/GFPmek_R product as template, 60dC

PCR purified the remaining 40ul from lane 8, and used 1ul in a new 50ul hisGFP_F_BB/GFPmek_R_BB PCR

Incubated membrane with anti-PDZ serum (#742, 1:250 in TBST/blocking buffer), 2h on shaker at room temp. Then washed 3x15min with TBST. Then incubated membrane with AlexaFluor 680 goat anti-rabbit (1:5000 in TBST/blocking buffer), 45min on shaker at room temp. Washed 2x10min TBST, 2x10min TBS, rinse with distilled water.

Lane 1: SeeBlue2 ladder

Lane 2: DC-1

Lanes 3-6: JLO159, -PDZ1, 2, 1+2, not induced

Lanes 7-10: JLO159, -PDZ1, 2, 1+2, induced

7/10/07

Ran 10ul colony PCRs of PDZ1, PDZ2, PDZ1+2 in pSB2K3, and of sLO, and of B0015, with 30 cycles (95dC 30s, 55dC 30s, 72dC 1m), in the grey 5096 and then loaded into an egel. But I only got two very light bands. Repeated the PCRs, picking from the streaks, switched to the machine by the 5088 entrance, and because of the Lpp29FS annealing temperature, I changed to 10 cycles of (95dC 30s, 45dC 30s, 72dC 1m) then 20 cycles of (95dC 30s, 55dC 30s, 72dC 1m). Then I got bands. So either the 45dC annealing or switching machines did the trick. I need to eventually do an experiment between the machines to see if the one in 5096 is faulty.

Lane 1: 1kb+

Lanes 2, 3: PDZ1 in pSB2K3, VF2/VR, #1 and 2

Lanes 4, 5: PDZ2 in pSB2K3, VF2/VR, #1 and 2

Lanes 6, 7: PDZ1+2 in pSB2K3, VF2/VR, #1 and 2

Lane 8: B0015, VF2/VR, #1 and 2

Lanes 9-11: sLO #1-3

Lane 12: GFPmek PCR, hisGFP_F_BB/GFPmek_R_BB, using PCR-purified product from "lane 8" on 7/6/07 as template (~30ul PCR, loaded 10ul)

TOPO cloned 4ul of the GFPmek PCR purified product from 7/6/07 and 4ul of 7/9/07 GFPmek PCR which used this as a template (not purified). added 1ul salt solution and 1ul TOPO vector. Transformed each into 30ul Top10 cells.

Also did a transformation of P0140, P0340, P0440 into 20ul Top10 cells.

Made liquid cultures of sLO, PDZ1, PDZ2, PDZ1+2 in pSB2K3, J04500, and B0015.