User:Perry/Summer 2006 Harvard iGEM work

6/12/06

Incubated DNA nanostructure folding interactions. DNA scaffold and oligonucleotides designed by Shawn Douglas. Protocol available here. Included 2 negative controls, one with water instead of scaffold, one with water instead of oligonucleotides.

Transformed R0010, E0241, and E7104 parts into 15 ul aliquots of chemically competent OneShot Top10 cells.

6/13/06

Ran gels from nanostructure incubations yesterday. 2% agarose gel, 130V, 45min. Loaded 10ul sample + 1ul dye, and 10ul 1kb ladder.

Lane 5: 1 kb ladder

Lane 6: DNA nanostructure (+)

Lane 7: (-) control with no oligonucleotides

Lane 8: (-) control with no DNA scaffold

Prepared 2 liquid cultures from each transformation, 5ml LB + 50ul of 5mg/ml ampicillin, shaken at 180rpm and 37dC overnight.

6/14/06

Retrieved liquid cultures (3x2). I accidentally dropped culture #2 from R0010 transf and spilled half the volume. Set aside 1ml of each culture for glycerol stock. Performed miniprep on remaining culture. Accidentally performed 2 washes with PE. Eluted with 50ul water.

The nanodrop gave negative readings of DNA concentration for the minipreps.

Prepared 25ul digest reactions with 8ul of both minipreps of R0010 (SpeI/PstI) and of E0241 (XbaI/PstI). Incubated 1 hour at 37dC held at 4dC, then incubated at 80dC for 20 min. Added 2ul loading dye to each digest mixture, then ran total mixtures in gel, 45 min, 130V.

Lane 8: 1 kb ladder

Lane 9: R0010, miniprep from culture #1, SpeI/PstI digested

Lane 10: R0010, miniprep from culture #2, SpeI/PstI digested

Lane 11: E0241, miniprep from culture #1, XbaI/PstI digested

Lane 12: E0241, miniprep from culture #2, XbaI/PstI digested

We got no bands, and so performed no gel extraction. We probably had a failed miniprep, evident from the negative number Lewis got from the nanodrop. I guess 2 PE washes is bad.

6/15/06

Using Tiffany and Matt's gel extracted vector/insert, we took 2ul of their vector and 6ul of their insert and performed a ligation. Then we transformed into Top10.

6/16/06

Observed two colonies on transformation plate which fluoresced green under microscope (probably due to leaky expression of GFP). Left plates in refrigeration.

6/19/06

Picked the two colonies from the transformation plate and prepared two 5ml inoculations with 50ul amp (5mg/ml).

6/20/06

Miniprepped two inoculations, eluting with 30ul water. Nanodrop read 98ng/ul for #1 and 73ng/ul for #2. We prepared 25ul XbaI/PstI digests of each, using 8ul DNA miniprep, incubating 90min 37dC, 15min 80dC. Then we ran the digests on a 1.2% e-gel, using 10ul digest + 10ul water. We also loaded 10ul 1X 1kb ladder + 10ul water.

Lane 1: 1kb+ ladder

Lane 5: XbaI/PstI digest of R0010-E0241 #1

Lane 6: XbaI/PstI digest of R0010-E0241 #2

6/22/06

Prepared sequencing reactions. Chris had ordered new primers, reconstituted at 20uM. I made a 1:10 dilution for 2uM.

PT001 8ul miniprep #1, 4ul VF2

PT002 8ul miniprep #1, 4ul VR

PT003 8ul miniprep #2, 4ul VF2

PT004 8ul miniprep #2, 4ul VR

7/3/06

Went to MIT to pick up plasmids with streptavidin clones from Mark Howarth at the Ting lab (Howarth et al, 2006).

• Streptavidin, wild-type, 0.1 ug/ul (StrepWT)
• Streptavidin, wild-type plus His6 tag (alive), 0.09 ug/ul (StrepH)
• Streptavidin, mutant (dead), 0.09 ug/ul (StrepD)

7/5/06

Ordered primers to PCR out the streptavidin clones.

Ting lab's sequences

Primers

Diluted plasmids 1:50, 1ul + 49ul water.

Transformed with OneShot Top10 chemically competent cells. Aliquotted 2x15ul for pUC19 (+) and for (-). Aliquotted 3x30ul for StrepWT, StrepH, and StrepD. Used 5ul of plasmid 1:50 dilutions and 2.5ul of pUC19. Put plates in incubator at 5:20pm.

7/6/06

Took plates out at 10:30am; they were overgrown. I had difficulty picking out single colonies. I streaked and inoculated liquid cultures from two colonies from each transformation. Liquid culture: 30ml LB + 300ul amp, 6x4.5ml aliquots. Put into incubation at 11:00am.

Returned at 1:00am to take out streak plate and to perform miniprep. Included PB step and eluted with 30ul water. The tubes for elution of minipreps from colonies StrepWT #1 and StrepH #2 cracked in the centrifuge. Prepared new 4.5ml liquid cultures from the streaked colonies; put in incubator around 2:00am.

7/7/06

Received StrepMF and StrepMR primers; reconstituted them in EB at 100uM. Then made another dilution to 5uM with 5ul of 100uM + 95ul EB. Did the same with OmpAF and OmpAR primers.

Nanodropped the StrepD minipreps, got a rather jagged curve. StrepD#1 was ~45ng/ul, StrepD#2 was ~25ng/ul. I made dilutions of 1:50 and 1:25 respectively in water.

Made a 1:2 dilution in water of the 1:50 dilution of original SA-dead (aka StrepD) from MIT (made 7/5/06), thus a final dilution of 1:100.

Prepared three PCR reactions. I tried to go for 50ul total with 45ul Platinum PCR Supermix, 200nM each primer, 1ng template DNA.

(0) 45ul Platinum PCR Supermix + 2ul StrepMF (5uM) + 2ul StrepMR (5uM) + 1ul SA-dead (1:100)

(1) " + 1ul StrepD#1 (1:50)

(2) " + 1ul StrepD#2 (1:25)

Incubated 95dC for 10min, 30 x (95dC for 1min, 55dC for 1min, 72dC for 1min), 72dC for 10min, 4dC forever.

Ran PCR reactions on 1.2% e-gel for 15min. 10ul of each + 10ul water, 10ul 1X 1kb ladder + 10ul water. Included a lane of 10ul 5uM StrepMR primer + 10ul water to test visualization of ssDNA with ethidium bromide.

Lane 1: 1kb ladder

Lane 2: PCR (0)

Lane 3: PCR (1)

Lane 4: PCR (2)

Lane 5: 1kb ladder

Lane 6: StrepMR primer

The StrepMR primer showed a visible band.

None of the PCR reactions were successful. After brief contemplation, I realized a very good reason why. StrepMF and StrepMR were oligos designed for mutagenesis and NOT as forward/reverse primers for PCRing the mutant StrepD.

However, it's confusing that there isn't even a primer dimer band. StrepMF/StrepMR are complementary over ~15 base pairs.

Removed liquid cultures (StrepWT#1, StrepH#2) from last night at 1:30pm. Pelleted and left in freezer over lunch. Completed miniprep upon return. Included PB step and eluted with 30ul water.

7/10/06

Made a 1:2 dilution in water of the 1:50 dilution of original StrepWT and StrepH from MIT (made 7/5/06), thus a final dilution of 1:100.

Nanodropped the minipreps for ng/ul, and made corresponding dilutions in water. StrepWT#1, 41.2, 1:40. StrepWT#2, 23.9, 1:20. StrepH#1, 21.3, 1:20. StrepH#2, 53.7, 1:50. StrepD#1, 44.9, 1:40, StrepD#2, 15, 1:15.

Received primers StrepF, StrepR, StrepFS, StrepRS, StrepHR, StrepHRS. Reconstituted in EB to 100uM, and then diluted 10 of 100uM with 90ul EB for a 10uM diluted stock. Also made 10uM diluted stock from 100uM stocks StrepMF and StrepMR received 7/7/06.

Prepared PCR reactions. Made a partial master mix (enough for 10 tubes) of 450ul Platinum PCR Supermix + 10ul StrepF(10uM) + 10ul StrepMF(10uM) + 10ul StrepMR(10uM). Aliquotted 45ul to 9 tubes as labeled below. Then added 1ul reverse primer and 1ul template DNA as appropriate. I then realized that I should have aliquotted 48ul instead of 45ul, and added 3 more ul of partial master mix to each tube; however, I ended up with an empty tube when I should have had ~45ul extra. Something possibly went wrong in aliquotting?

All primers at 10uM. All template DNA ~1ng/ul.

W0: (45ul Platinum PCR Supermix + 1ul StrepF + 1ul StrepMF + 1ul StrepMR) + 1ul StrepRS + 1ul StrepWT plasmid (1:100 from MIT)

W1: ( ) + 1ul StrepRS + 1ul StrepWT#1 miniprep(1:40)

W2: ( ) + 1ul StrepRS + 1ul StrepWT#2 miniprep(1:20)

H0: ( ) + 1ul StrepHRS + 1ul StrepH plasmid (1:100 from MIT)

H1: ( ) + 1ul StrepHRS + 1ul StrepH#1 miniprep(1:20)

H2: ( ) + 1ul StrepHRS + 1ul StrepH#2 miniprep(1:50)

D0: ( ) + 1ul StrepRS + 1ul StrepD plasmid (1:100 from MIT)

D1: ( ) + 1ul StrepRS + 1ul StrepD#1 miniprep(1:40)

D2: ( ) + 1ul StrepRS + 1ul StrepD#2 miniprep(1:15)

Put into incubator with PCR program. Modified from an initial 95dC for 10min with hot start, to initial 95dC for 15min without hot start. (I got annoyed with having to go back to the PCR machine to make it continue from pause.)

Took 10ul from each PCR and of 1X 1kb+ ladder, mixed with 10ul water, and loaded into 1.2% e-gel.

Lane 1,11: 1kb+ ladder

Lanes 2-4: W0,1,2

Lanes 5-7: H0,1,2

Lanes 8-10: D0,1,2

Lane 12: Water

Ug, no bands of the expected 1kb size. There is a uniform band around 100bp, most probably a primer dimer.

Sigh, time for troubleshooting.

Prepared new 3ml liquid cultures from streak plates of W,H,D-1,2.

7/11/06

Received E. coli K12 genomic DNA (5ug) from ATCC. Rehydrated in 100ul water (for 50ng/ul) and incubated at 37dC for one hour, as directed.

Repeated nine PCRs from yesterday, but using 1ul of 1:50 plasmid from MIT and undiluted minipreps. Included one PCR reaction of 1:50 StrepW (plasmid from MIT) with water replacing the StrepMF and StrepMR primers, and one PCR reaction with 2ul OmpAF(5uM), 2ul OmpAR(5uM), and 2ul K12 genome(50ng/ul).

Ran e-gel, loading each well with 10ul PCR mixture/ladder + 10ul water.

Lane 1: 1kb+ ladder

Lanes 2-4: W0,1,2

Lanes 5-7: H0,1,2

Lanes 8-10: D0,1,2

Lane 11: W0 without StrepMF/StrepMR

Lane 12: OmpA

The Strep bands are more significant. Same primer dimers around 100, but then there's an expected weak band in lanes 2-10 at around 400, but a strong band in lane 11. There's also another weak band around 300. I believe that the mutagenesis primers are screwing up the efficiency of the PCR. There's no visible band for OmpA in lane 12.

Performed PCR purification of W0 without StrepMF/StrepMR and of OmpA PCRs.

Performed agarose gel electrophoresis of W,H,D-0,1,2 (poor loading of lanes 3 and 7).

Lanes 1,11: 1kb+ ladder

Lanes 2-4: W0,1,2

Lanes 5-7: H0,1,2

Lanes 8-10: D0,1,2

I forgot to take a picture before I excised 400bp bands from W2, H2, and D1 and disposed of the gel.

Prepared PCR reactions of W,H,D0 without StrepMF/R. Also prepared a 2nd PCR reaction with 1ul PCR-purified OmpA, and a 1st PCR reaction for OmpA using 10ul of K12 genomic DNA (50ng/ul, so 500ng), filling to a 60ul PCR mixture, with 2.4ul of OmpAF and OmpAR (5uM).

Removed liquid cultures of W,H,D-1,2 and refrigerated.

7/12/06

Performed gel purification of W2, H2, D1 gel slices from yesterday. Have they been successfully mutated to lose the XbaI site? We'll have to see.

Ran 1.2% e-gel of PCRs.

Lane 1: 1kb+

Lane 2,3,4: W0,H0,D0 without StrepMF/R

Lane 5: OmpA (a 2nd PCR of PCR-purified OmpA)

Lane 6: OmpA (a 1st PCR of 500ng K12 genomic DNA)

Performed PCR purifications of W0,H0,D0 without StrepMF/R and OmpA (2nd PCR).

Performed TOPO cloning with 4ul of gel-purified W2,H2,D1 (undetermined mutagenesis) and PCR-purified W0,H0,D0 (no mutagenesis) and OmpA (2nd PCR), 1ul salt solution, 1ul TOPO vector. Transformed each with 20ul Top10 chemically competent cells, including a (+) with 2.5ul pUC19 and a (-) with nothing.

I talked to Chris and realized that I've been going about mutagenesis completely wrong-ly. So I redid the mutageneses right-ly. Prepared six PCR reactions (all primers 10uM):

• 47ul Platinum PCR Supermix + 1ul StrepF + 1ul StrepMR + 1ul StrepW,H,D (1:50 plasmids from MIT)

• " + 1ul StrepRS + 1ul StrepMF + 1ul StrepW,D

• " + 1ul StrepHRS + 1ul StrepMF + 1ul StrepH

PCR purified the mixtures. Then using those, I prepared three PCRs

• StrepW(mut) = 45ul Platinum PCR Supermix + 2ul StrepW(F/MR) + 2ul StrepW(RS,MF)

• StrepH(mut) = " + 2ul StrepH(F/MR) + 2ul StrepH(HRS,MF)

• StrepD(mut) = " + 2ul StrepD(F/MR) + 2ul StrepD(RS,MF)

After 10/30 cycles, I paused the PCR machine and added 1ul StrepF and 1ul StrepRS to StrepW and StrepD, 1ul StrepF and 1ul StrepHRS to StrepH. Then I restarted the PCR machine to finish the remaining 20 cycles.

7/13/06

I took out the plates from the TOPO cloning/transformation; got a lot of colonies on all plates except (-) which had no colonies. I threw out the plates transformed with TOPO clone of gel-purified W1,H1,D2, and I threw out those gel-purified samples as well (because they had come from an incorrect PCR in which I mixed both forward/reverse and mutagenesis primers).

I streaked and inoculated 3ml cultures (30ul of 5mg/ml ampicillin) from the StrepW,H,D (no mut) and full OmpA transformations. Put in incubation around 2:00pm.

PCR purified StrepW,H,D(mut) from yesterday.

Ran 1.2% e-gel of the PCR purifications from yesterday and today.

Lanes 1,11: 1kb+ ladder

Lanes 2-4: StrepW(F/MR),(RS/MF),(mut)

Lanes 5-7: StrepH(F/MR),(HRS/MF),(mut)

Lanes 8-10: StrepD(F/MR),(RS/MF),(mut)

Topo cloned/transformed StrepW,H,D(mut). Aliquotted 15ul of Top10 chemically competent cells for the transformations, 7ul for (+) and 7ul for (-). Put in incubation around 2:00pm.

7/14/06

Took StrepW,H,D(no mut) and full OmpA liquid cultures out at 8:00am. Pelleted.

Took StrepW,H,D(mut) plates out; got a lot of colonies. Streaked and inoculated 3ml liquid cultures around 8:30am.

Miniprepped StrepW,H,D(no mut) and (mut), and full OmpA. Included PB step and eluted with 40ul water.

Prepared seven 20ul digests. First made a master mix of 20ul Buffer2, 20ul water, 4ul XbaI, 4ul PstI, 2ul BSA(100X), enough for 10. Aliquotted 5ul of master mix to each tube, then added 15ul of miniprep to each tube. Incubated at 37dC for 12h, 80dC for 20min, 4dC forever.

Received single-chain dimeric streptavidin from Dr. Filiz Aslan, as dry samples in tubes: SCD-NM (not mutant), C2, E2. She told me there was about 100ng in each tube. Reconstituted in 50ul EB for 2ng/ul, then made a 20ul stock, 1:2, for 1ng/ul.

Transformed SCD-NM, C2, E2, using 20ul Top10 OneShot chemically competent cells and 1ul of DNA (1ng/ul). Left in incubation at 1:30am.

7/15/06

Took out transformation plates; got a lot of colonies. Streaked and inoculated 3ml cultures from one colony from each plate: SCD-NM, C2, E2, at around 4:30pm.

Ran XbaI/PstI digests on 1% agarose gel.

Lane 1: 1kb+ ladder

Lanes 3,7,11: StrepW,H,D(no mut)

Lanes 5,9,13: StrepW,H,D(mut)

Lane 15: full OmpA

The 300bp/120bp bands in lanes 3, 7, 11 are the segments of the streptavidin BioBrick which have been cleaved by XbaI because of the XbaI site at bp#120 in the sequence. The 400bp band in lanes 5, 9, 13 is the full streptavidin BioBrick, not cleaved by XbaI because of the mutagenesis removing the XbaI restriction site. The 50bp band in all the lanes is the segment of the TOPO vector from the BioBrick PstI site to an XbaI site 50bp downstream. The 3kb/1kb bands in all the lanes are the segments of the rest of the TOPO vector, cleaved by XbaI, I think.

Excised the 400bp bands from lanes 5, 9, 13, and 1kb band from lane 15.

I just realized the futility of excising the OmpA 1kb band. First, that band is mixed with the 1kb band from the Topo vector (there is an XbaI site 1kb upstream of the insertion site). Second, I don't need a digested OmpA because I will not be using the full version.

7/16/06

Miniprepped SCD-NM, C2, E2 at around 8:30am. Included PB step and eluted with 30ul water.

7/17/06

Rec'd primers and reconstituted all in EB to 100uM, then made a dilution of 5ul of 100uM + 95ul EB, for 5uM.

Prepared 11 PCR reactions. Used SCD-NM, C2, E2 2ng/ul reconstitutions from 7/15/06, OmpA miniprep from 7/14/06, and 5uM primers. Also prepared 1 PCR reaction to check for OmpA insertion into the Topo vector using M13 forward and reverse primers at 20uM.

S, C, E(F/R): 45ul Platinum Supermix HiFi + 2ul StrepSCDF + 2ul StrepSCDRS + 1ul S, C, E

S, C, E(F/MR): " + 2ul StrepSCDF + 2ul StrepSCDMR + 1ul S, C, E

S, C, E(MF/R): " + 2ul StrepSCDMF + 2ul StrepSCDRS + 1ul S, C, E

O66: " + 2ul OmpA46F + 2ul OmpA66R + 1ul OmpA

O159: " + 2ul OmpA46F + 2ul OmpA159R + 1ul OmpA

OM13: " + 0.5ul M13F + 0.5ul M13R + 3ul water + 1ul OmpA

Sent out sequencing reactions to Genewiz, using the StrepW,H,D(mut) and OmpA minipreps from 7/14/06 (depleted the supply).

PT005: 8ul StrepW(mut), M13F(-21) to be added

PT006: 8ul StrepW(mut), M13R to be added

PT007: 8ul StrepH(mut), M13F(-21) to be added

PT008: 8ul StrepH(mut), M13R to be added

PT009: 8ul StrepD(mut), M13F(-21) to be added

PT010: 8ul StrepD(mut), M13R to be added

PT011: 8ul OmpA, M13F(-21) to be added

PT012: 8ul OmpA, M13R to be added

Ran a 1% agarose gel of the PCR reactions. Added 5ul loading dye, then loaded 40ul into wells.

Lane 1, 14, 15: 1kb+ (or at least I thought)

Lanes 2-4: SCD-NM MF/R, F/MR, F/R

Lanes 5-7: C2 MF/R, F/MR, F/R

Lanes 8-10: E2 MF/R, F/MR, F/R

Lane 11: OmpA ompA46F/ompA66R

Lane 12: OmpA ompA46F/ompA159R

Lane 13: OmpA M13F(-20)/M13R

Excised ~300bp band from lanes 2, 5, 8; ~800bp band from lanes 3, 6, 9; ~1kb band from lanes 4, 7, 10; ~150bp band from lane 11; ~400bp band from lane 12.

Performed gel extraction. Included extra QG wash and eluted with 30ul EB.

Ran a PCR with 45ul PCR Supermix HiFi + 2ul S(MF/R) gel extraction + 2ul S(F/MR), " + C(MF/R) + C(F/MR), " + E(MF/R) + E(F/MR). After cycle 10, I added 2ul StrepSCDF (5uM) and 2ul StrepSCDRS (5uM) to each reaction.

7/18/06

Rec'd LppFS, Lpp29R, Lpp78R, Lpp78RS primers. Reconstituted in EB to 100uM, then made a dilution to 5uM.

Prepared three PCR reactions, primers at 5uM.

Lpp29: 45ul Platinum PCR Supermix HiFi + 2ul K12 genomic DNA (50ng/ul) + 2ul LppFS + 2ul Lpp29R

Lpp78: " + " + " + 2ul Lpp78R

Lpp78S: " + " + " + 2ul Lpp78RS

Ran 1% agarose gel of PCRs (made gel larger than usual, 120ml TBE and 1.2g agarose). Added 5ul loading dye to each reaction and loaded 40ul. 130V, 50min.

Lane 1: 1kb+

Lane 2: SCD-NM(mut)

Lane 3: C2(mut)

Lane 4: E2(mut)

Lane 5: Lpp29

Lane 6: Lpp78

Lane 7: Lpp78S

Cut out bands from lanes 5, 6, 7. Gel extracted Lpp29 and Lpp78S.

Prepared six PCRs using yesterday's gel-extracted S, C, E (F/R) as template.

45ul Platinum PCR Supermix HiFi + 2ul StrepSCDMF + 2ul StrepSCDRS + 1ul S,C,E(F/R)

45ul Platinum PCR Supermix HiFi + 2ul StrepSCDF + 2ul StrepSCDMR + 1ul S,C,E(F/R)

Ran PCR reactions on 120ml 1% agarose gel (added 5ul loading dye, loaded 40ul). 130V, 50min

Lane 1: 1kb+

Lanes 2,4,6: S, C, E (MF/R)

Lanes 3,5,7: S, C, E (F/MR)

Cut out ~250bp bands in lanes 2, 4, 6, and ~650bp bands in lanes 3, 5, 7. Gel extracted. Included extra QG step and eluted with 30ul EB.

Topo cloned/transformed using 4ul of gel extractions of Lpp29, OmpA66, and OmpA159, and 20ul Oneshot Top10 competent cells. Left in incubator at 3:00am.

7/19/06

Ran S, C, D(mut) on 120ml 1% agarose gel.

Lane 1: 1kb+

Lane 2, 3, 4: S, C, E(mut)

Cut out the ~350bp bands and gel extracted. Included extra QG step and eluted with 30ul EB.

Sent these gel extractions out for sequencing.

PT013: S(mut), StrepSCDF

PT014: S(mut), StrepSCDRS

PT015: C(mut), StrepSCDF

PT016: C(mut), SrepSCDRS

PT017: E(mut), StrepSCDF

PT018: E(mut), StrepSCDRS

Prepared eighteen new PCR reactions using PCR Supermix, NOT High Fidelity. (all primers at 5uM)

P1: 22ul PCR Supermix + 1ul StrepSCDF + 1ul StrepSCDMR + 1ul S, C, E (from 2ng/ul plasmid reconstitution 7/14/06)

G1: " + " + " + 1ul S, C, E (F/R) gel ex from 7/17/06

P2: 22ul PCR Supermix + 1ul StrepSCDRS + 1ul StrepSCDMF + 1ul S, C, E (recon)

G2: " + " + " + 1ul S, C, E (gel)

S, C, E (mut): 22ul PCR Supermix + 2ul S,C,E(F/MR) + 2ul S,C,E(R/MF) (gel ex 7/18/06)

Primer control 1: 22ul PCR Supermix + 1ul StrepSCDF + 1ul StrepSCDMR

Primer control 2: 22ul PCR Supermix + 1ul StrepSCDRS + 1ul StrepSCDMF

Primer control F/R: 22ul PCR Supermix + 1ul StrepSCDF + 1ul StrepSCDMRS

After 11 cycles (was supposed to be 10), I paused the machine, took out S, C, E (mut) and added 1ul StrepSCDF and 1ul StrepSCDRS to each, replaced them, and continued the PCR incubation.

Ran 1% 100ml agarose gel, 20 wells.

Lanes 1,20: 1kb+

Lanes 2,4,6: S,C,E(F/MR) (plasmid)

Lanes 3,5,7: S,C,E(R/MF) (plasmid)

Lanes 8,10,12: S,C,E(F/MR) (gel ex)

Lanes 9,11,13: S,C,E(R/MF) (gel ex)

Lanes 14-16: S,C,E(mut)

Lanes 17-19: Primer controls 1, 2, F/R

Made cuts around all visible bands in lanes 2-13, and left in 4dC overnight.

At around 7:00pm, I took out the Lpp29, OmpA66, and OmpA159 Topo cloning/transformation plates. I streaked and inoculated 3.3ml (33ul ampicillin, 5mg/ml) liquid cultures from one colony of each transformation. There weren't any carbinicillin plates, so I used a kanamycin plate; the Topo vector also carries kanamycin resistance.

7/20/06

Performed miniprep of Lpp29, OmpA66, OmpA159. Included PB step and eluted in 40ul water.

Prepared three digests. Master mix of 6ul water, 6ul Buffer 2, 1.2ul XbaI, 1.2ul PstI, 0.6ul BSA(100X). 15ul miniprep Lpp29,OmpA66,OmpA159 + 5ul master mix. Incubated at 37dC for 2h, 80dC for 20min.

Took out cut bands. 650bp from lanes 2,4,6: S1, C1, E1. 250bp from lanes 3,5,7: S2, C2, E2. Included extra QG step and eluted in 20ul EB.

Prepared three PCR reactions: 20ul Platinum PCR Supermix + 2ul S1,C1,E1(gel ex) + 2ul S2,C2,E2(gel ex). Paused after 10 cycles and added 1ul StrepSCDF and 1ul StrepSCDRS.

Ran 100ml 1% agarose gel, 130V, 45min, but the gel came out awful due to buffer that's been reused a few too many times.

Got sequences back for StrepW, H, D(mut). StrepW has a perfect sequence 1-444 from both M13F and M13R sequencing reactions. StrepH has a mutation at bp#344 T to C, which in the reading frame is a GCT to GCC, a silent mutation for Alanine. Strep D has a perfect sequence 1-409 from M13R reaction, and a perfect sequence 410-444 from M13F reaction.

Prepared StrepW,H,D(mut) cultures for midiprep in 100ml Terrific broth + 100ul kanamycin (50mg/ml). Also prepared new cultures of Lpp29, OmpA66, and OmpA159, 3ml LB + 30ml amipcillin (5mg/ml). Put in incubator at 6:30pm.

7/21/06

Removed liquid cultures at 10:30am.

Repeated PCR Reactions and digests from yesterday. Ran 1% 100ml agarose gel.

Lane 1,8: qkb+

Lanes 2-4: S,C,E(mut)

Lane 5: Lpp29 XbaI/PstI

Lane 6: OmpA66 XbaI/PstI

Lane 7: OmpA159 XbaI/PstI

Performed miniprep on Lpp29, OmpA66, OmpA159, including PB step and eluting in 30ul water. Sent off these minipreps for sequencing, 8ul for each reaction.

PT019: Lpp29, M13F(-21)

PT020: Lpp29, M13R

PT021: OmpA66, M13F(-21)

PT022: OmpA66, M13R

PT023: OmpA159, M13F(-21)

PT024: OmpA159, M13R

Performed midiprep on StrepW, StrepH, StrepD. Used N3 instead of P3, and skipped step 8, the 2nd centrifugation. Mixed more 70% ethanol (70ml of 95% ethanol + 25ml water).

7/24/06

Prepared liquid cultures (2ml LB + 20ul ampicillin 5mg/ml) of StrepW, StrepH, StrepD, and full OmpA, for glycerol stocks tomorrow.

Nanodropped StrepW, StrepH, StrepD midipreps. All 500-600 ng/ul.

Prepared three double digests. 0.3ul BSA(100X), 0.6ul XbaI, 0.6ul PstI, 2ul StrepW/H/D midiprep, 3ul Buffer2, 23.5ul water. Incubated at 37dC for 12h, 80dC for 20min.

Prepared three PCRs. 45ul PCR Platinum Supermix, 2ul StrepSCDF(5uM), 2ul StrepSCDRS(5uM), 1ul S/C/E(2ng/ul reconstitution from 7/14/06). PErformed PCR purification, eluting with 30ul water. Then prepared three digests: 0.35ul BSA, 0.7ul PstI, 3.5ul Buffer3, 30ul PCR-purified S/C/E. Incubated at 37dC for 8h, 80dC for 20min.

7/25/06

Made glycerol stocks of StrepW, StrepH, StrepD, full OmpA, with 1ml liquid culture + 666ul 50% glycerol. Froze in screwcap tubes at -80dC.