Team:SJTU-BioX-Shanghai/Notebook/log

July 1st, 2012

Plasmid Extraction

Fig7.1 Digestion results

dsbASS, lactamase, lgt, GFP, FL3, S1P, S2P, pETDuet-1, pACYLDuet-1, pRSFDuet-1, PBAD-1

3 tubes each, 11*3=33 tubes altogether.

pBAD, S1P, S2P needs to be extracted again.

Digestion Identification

Restriction enzyme: EcoRI, PstI

Only lactamase 1-2, GFP1-2 have correct bands

Pick colonies of dsbASS, lgt and FL3 again and incubate in 2ml EP tubes(*10).

July 2nd, 2012

Tailing and Colony Picking

pET, pRSF, pACYC, pBAD, vioD, vioE

Each pick 4 colonies.

PCR

Fig7.2 PCR results

lgt*7, dsb*7, FL3*7, S1P*5, S2P*3

vioA, vioB, vioC, vioD, vioE

Plasmid Extraction

vioABCE

Incubation

S1P-1, S1P-2, S2P-1, S2P-3, dsbASS-6, dsbASS-1, FL3-6, FL3-3, FL3-7, 10 tubes altogether

10ul into 5ml EP tube, incubated in 37℃.

Transformation

Transform PDZ(5.2ng/ul), GBD(3.3ng/ul) and SH3(7.7ng/ul) 1ul each into 30ul competent cells.

Transform vioABCE and vioABDE(Kn resistant) 0.5ul each into 50ul competent cells.

July 3rd, 2012

PCR

Fig7.3 Diegestion results

dsbASS(bacteria), site1 and site2(plasmid)

Incubation

Pick colonies from the plates (PDZ, GBD, SH3, vioABCE and vioABDE). Incubated from 9:00am for 24h.

Incubate pACYC and pRSF(1ul into 5ml)

Pasmid Extraction

pET 1-4, pBA 1-4, vioD 1-3, vioE 1-4

FL3 1-7, S1P 1-2, S2P 2-3, vioABCE 1, vioABCE 2

Digestion identification

Restriction enzyme: EcoRI, PstI

July 4th, 2012

Fig7.4.1 PCR results

Fig7.4.2 Gel extraction

Pasmid Extraction

pACYC, pRSF

PCR and Gel Extraction

dsbAss, lgt, FL3 (3-step method)

Only FL3 has band.

Colony Picking

vioABCE, vioABDE, GFP, lactamase

July 5th, 2012

Fig7.5 PCR and tailing results

Tailing

FL3

PCR

dsbASS, lgt

(KOD, 2-step method)

Ligation and Transformation

Ligate FL3 with T vector. (Solution I system)

2ul ligation product was transformed into 50ul DH5a, coated 2 plates(Amp)

July 6th, 2012

Fig7.6 PCR and digestion results

Digestion identification

lgt and dsbASS

PCR

lgt and dsbASS

July 7th, 2012

Colony Picking

Plates: FL3-T-1, FL3-T-2(coated on July 5th)

Pick 3 colonies from each plates

Incubate

Incubated at 37℃ from 9:30am

July 8th, 2012

Fig7.8 FL3 plasmids

Transformation

GWPT1 and GVPT1, each 1ul, were transformed into 50ul competent cells, coated 2 plates(Kn)

GW334, JD504and JD505, each 2ul, were transformed into 30ul competent, coated 3 plates(Amp)

Pasmid Extraction

FL3-1 and FL3-2, 3 tubes each

Digestion identification

Restriction enzyme: EcoRI, PstI

No objective sequences

July 9th, 2012

Fig7.9 pACYC, pET and pRSF plasmids

Colony Picking

VVD, JD504, JD505, GW334 in the morning, pick 2 colonies from each plate.

lgt in the afternoon, pick 3 colonies.

Incubate at 240rpm for 20h.

Plasmid Extraction

pACYC-1(26.6ng/ul), pACYC-2(17.7ng/ul), pACYC-3(27.0ng/ul)

pET-1(26.1ng/ul), pET-2(16.8ng/ul), pET-3(28.7ng/ul)

pRSF-1(17.1ng/ul), pRSF-2(30.2ng/ul), pRSF-3(9.9ng/ul)

The size of DNA fragments may be incorrect.

July 10th, 2012

Fig7.10.1 Digestion identification of vectors and lgt-T

Plasmid Extraction

lgt1-1(71.6ng/ul), lgt1-2(48.2ng/ul), lgt2-1(183.2ng/ul), lgt2-2(131.3ng/ul)

Each has 2 bands and high brightness.

Digestion Identification

pSRF, pET and pACYC cut by HpaI

lgt-T cut by EcoRI and PstI

PCR and Gel Extraction

Fig7.10.2 PCR result

FL3(27.3ng/ul), dsbASS(18ng/ul), lgt(26.8ng/ul)

July 11th, 2012

Fig7.11 Ligation products

Tailing

dsbA11(91.7ng/ul), dsbA22(91.7ng/ul), FL311(52.6ng/ul), FL322(59.7ng/ul), lgt11(8.7ng/ul), lgt22-1(21.9ng/ul), lgt22-2(41.4ng/ul)

The size of the bands was correct

Ligation

System: 5ul solutionI + 0/5ul T-vector + 4.5ul DNA

dsbASS-lac, FL3-lgt, lgt-site1, lgt-site2

Begin at 16:00pm

Plasmid Extraction

pACYC-2(88.2ng/ul), pACYC-3(127.1ng/ul), pACYC-4(108.4ng/ul)

pET-1(80.0ng/ul), pET-2(138.2ng/ul), pET-4(123.9ng/ul)

pRSF-3(109.4ng/ul), pRSF-4(107.0ng/ul)

Digestion Identification

Restriction enzyme: HpaI

pRSF-3&4, pACYC-2&3&4, pET-1&2&4, siteI(pBAD),site2(2-1E)

July 12th, 2012

Fig7.12 PCR results

PCR

Bacteria solution: dsb-1(1-8), dsb-2(1-8), lgt-1(1-8), lgt-2(1-8), 0.5ul each

KOD, 3-step method

Successful PCR products: dsb*3, lgt*3

Incubate the correct bacteria(dsb*3, lgt*3) overnight

July 13th, 2012

Fig7.13.1 PCR results

Fig7.13.2 Digestion Identification

PCR

FL3

KOD, 3-step method

Digestion Identification

Restriction enzyme: EcoRI, SpeI

lgt, dsbASS, FL3

July 14th, 2012

Fig7.14 Digestion identification

Purification

PCR products of FL3

Digestion Identification

Restriction enzyme: EcoRI, XbaI

lac, lgt, GFP

Plasmid Extraction

dsbASS*3, GFP*2, lac*2, lgt*3

Preserve the strains.

Ligation

FL3-GFP, FL3-lgt

System: 5ul solutionI + 0.5ul GFP/LGT + 4.5ul FL3

July 15th, 2012

Fig7.15.1 PCR results(FL3-GFP)

Fig7.15.2 PCR results(FL3-lgt)

Colony Picking

Four plates: FL3-GFP*2, FL3-lgt*2

Pick 15 colonies from each plate

PCR

Bacteria solution(FL3-GFP, FL3-lgt)

Result: no bands

July 16th, 2012

Fig7.16 Digestion results

Digestion Identification

FL3 cut by EcoRI/SpeI

GFP and lgt cut by EcoRI/XbaI

System: 20ul (Takara)

Gel Extraction

Denatured under 65℃ first

July 17th, 2012

Ligation

FL3-GFP, FL3-lgt

July 18th, 2012

Fig7.18.1 PCR results

Colony Picking

lgt2(16 colonies)

FL3-GFP, FL3-lgt

Begin at 10:00am

PCR

lgt

KOD, 3-step method

Fig7.18.2 Digestion identification

Digestion Identification

Restriction enzyme: EcoRI/PstI

Ligation products: FL3-GFP, FL3-lgt

System: 20ul

July 19th, 2012

Fig7.19 Digestion Identification of lgt

Plasimid Extraction and Digestion Identification

lgt-8(85.3ng/ul), lgt-9(90.4ng/ul), lgt-11(110.5ng/ul)

Restriction enzymes: E/P, E/X

Result: unsuccessful reverse ligation

Ligation

Digestion: EcoRI,PstI, begin at 13:00

Denatured at 65℃

Ligation system: pMD18-T 0.5ul + dsbASS 1ul + Solution I 5ul + ddH2O

Transformation

dsbASS-T, GFP-T

July 20th, 2012

Fig7.20 Plasmids

Plasmid Extraction

pET-1: no bands

pET-2: incorrect sequence

vioABDE: no bands

Colony Picking

S1S2pET, S1S2pRSF

Digestion

S1S2pET and S1S2pRSF cut by BsgGI/XhoI(11:00)

July 21st, 2012

Fig7.21.1 Digestion results

Plasmid Extraction

pET: no bacteria growing

pACYC; correct

Digestion

S1S2pACYC cut by BsgGI/XhoI

S1S2pACYC, S1S2pRSF, S1pET and GFP-T cut by EcoRI/PstI (recycled)

PCR

Fig7.21.2 PCR results

site1-F and site1-R: correct

site2-F and site2-R: correct

Ligation

GFP 2ul + Plasmid 1ul + Solution 3ul

site2 0.5ul + S1pET 2.5ul + Solution 3ul

For 4 hours

Transform

50ul Top10 competent cells + 2.5ul ligation product

July 22nd, 2012

Fig7.22 Digestion results

Plasmid Extraction

Digestion

dsbASS-T, GFP-T-1, GFP-T-2

PCR

to test whether dsbASS and T vector have been ligated

Taq, 1ul template

Transformation

Ligation product: lgt-T

July 23rd, 2012

Fig7.23.1 PCR results(lgt)

PCR

a. identification of lgt

b. identification of GFP-Plasmid

July 24th, 2012

Fig7.24.1 Digestion results(E/X,E/P)

Digestion

Restriction enzymesL EcoRI/PstI, EcoRI/XbaI

Plasmid: lgt

Result: E/P positive, E/X negative

Construction

lac, lgt-2: EcoRI/SpeI

lgt-1, GFP: EcoRI/XbaI

Gel extraction: lac(6ng/ul),lgt2(12.4ng/ul), lgt1(30.8ng/ul, GFP(4.2ng/ul)

Ligation(6h): 1.5ul GFP-T + 3.5ul lgt2 + 5ul Solution I, 0.5ul lgt1 + 4.5ul lac + 5ul Solution I

PCR

Identification of dsbASS-T

July 25th, 2012

PCR

L-L, L-G

Primers: (lac-F,lgt-R), (lgt-F,GFP-R)

Taq, annealing temperature 56℃

Result: correct bands

Colony Picking

Pick corresponding L-L, L-G colonies and incubate for 12-24h

Purification

L-L(30.0ng/ul), L-G(25.5ng/ul)

Digestion

dsbASS cut by EcoRI/PstI

GFP-pRSF cut by EcoRI/XbaI

July 26th, 2012

Plasmid Extraction and Digestion

Plasmid: L-L, L-G

L-L cut by EcoRI/PstI(2h)

L-G cut by EcoRI/XbaI(2h)

Ligation

System: 4.5ul lac + 0.5ul lgt-GFP + 5ul Solution I

July 27th, 2012

Fig7.27.1 Plasmid(S1S2@pACYC,pET,pRSF)

Fig7.27.2 Plasmid(L-L1-6,L-G1-3)

Plasmid Extraction

S1S2 at pACYC, pET, pRSF

L-L 1,2,3,4,5,6

L-G 1,2,3

July 28th, 2012

PCR

July 29th, 2012

Fig7.29.1 Digestion results

Plasmid Extraction

pRSF *2

Digestion

lac-dsbASS

lac *4

Purification

PCR product: dsb-F + overlap-R

July 30th, 2012

Fig7.30 PCR results

Ligation

a.pACYC-S1GFP, b.pET-S1GFP, c.pRSF-S1GFP,d.pUC19-GFP(FL3),e.pUC19-lgt(FL3),f.pACYC-S2GFP

PCR

rbs-dsb-lac *8

FL3-GFP *4 (GFP-T)

FL3-lgt *4 (lgt-Plasmid)

Transformation

S1S2pET-S1GFP, S1S2pRSF-S1GFP, FL3-GFP

FL3-lgt, pUC19(1.5ul), dsb-lac

July 31st, 2012

Fig7.31 Digestion identification of the plasmids

Plasmid Extraction

lgt *2

lgt-GFP *4

Digestion Identification

Result: unsuccessful conncetion

Transformation

lac-pUC19

lgt-GFP-pUC19