Team:Peking/Notebook/JYJiao

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Contents

• July, 2010

• August, 2010

• September, 2010

• October, 2010

July

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7.04

positive transformation of the parts which will be used in the future: R0080(Para), B0032(RBS), K145001(T7polymerase) and B0015(Terminator).

7.05

1 pick two clones each plate and shave at 37℃ to amplify the bacteria

2 The first challenge: we don’t have the sequence of the plasmid PSD-MBD(containing the lpp-ompa-mbp) offered by Summers. After reading papers for the detailed information of this plasmid, I selected and ordered the sequence primers.

3 Miniprep of the plasmid.

4 DNA overnight double digestion of the plasmids.

20uL reaction system:

Buffer2 2uL

SpeI 1.5uL

PstI 1.5uL

RBS 5uL

ddH2O 10uL

Buffer3 2uL

XbaI 1.5uL

PstI 1.5uL

T7polymerase 5uL

ddH2O 10uL

Buffer 2uL

EcoRI 1.5uL

XbaI 1.5uL

terminator 5uL

ddH2O 10uL

7.06

1 Electrophoresis enzyme digestion reaction system in 1.5% agarose gel.

2. Excise the gel slice and extract the fragments.

3 ligation of insert DNA fragments into plasmid vector DNA for 1 hour

T7polymerase(P/X) 6uL

RBS-vector (P/S) 2uL

10x ligation buffer 1uL

T4 DNA Ligase 1Ul

4 transform the products of ligation and E0240

5.pick three clones and shake in 37℃ for 8h.

6 Miniprep of the E0240 and double digestion(E,X) in 37℃

7.07

1 Electrophoresis the enzyme digestion reaction system in 1.5% agarose gel.

2. Purify the fragments E0240(E X).

3 miniprep of the plasmid of rbs-T7polymerase-PSB1A2

4 double digestion rbs-T7polymerase-PSB1A2(X,P)and send to sequence to verify the plasmid.

5 Electrophoresis enzyme digestion reaction system in 1.5% agarose gel.

6 Excise the gel slice and extract the fragments of rbs-T7polymerase (insert).

7 ligation the rbs-T7polymerase to the vector with Para.

7-08

1 transformation the Para rbs-T7poly

2 the sequence results showed that rbs-T7polymerase is right but there is no result of OMPA-MBP.

7-09

1 pick several clones and amplify the bacteria.

2 miniprep the Para- rbs-T7poly

3 enzyme digestion of Para-rbs-T7poly (E,P) to verify

7-10

1. Electrophoresis enzyme digestion reaction system in 1.5% agarose gel. But the results is not right.

2. Ligation again.

3. transformation the Para rbs-T7poly

4. get the sequence of PSD-MBD, and it is right!

7-11

1. miniprep the Para-rbs-T7poly.

2. Enzyme digestion to verify

7-12

1 Electrophoresis enzyme digestion reaction system in 1.5% agarose gel. But the results is not right.

2 give up the arabinose promoter.

7-13

1 the PCR primer is arrived and begin to get the lpp-omp-mbp sequence with PCR.

SuperMix(trans) 10uL

Commercial For(with NdeI site) 1uL

Commercial Rev(with XhoI site) 1uL

Template plasmid P 0.5uL

ddH2O up to 20 uL

SuperMix(trans) 10uL

Standard For(with prefix) 1uL

Standard Rev(with suffix) 1uL

Template plasmid P 0.5uL

ddH2O up to 20 uL

2 Electrophoresis PCR reaction system in 1.5% agarose gel. Since the MBP is a tandem structure, the reverse primer can pair with the template at two different places, there are two bands. The larger band is selected and purified.

3 Enzyme digestion of Standard fragmets with XbaI and PstI and the Commericl fragments with NdeI and XhoI.

4 another pair of primers for PSD-MBD is ordered.

7-14

1 to verify whether the band purified from PCR is right, the fragments are digested with BamHI ,which only exists in the right fragments.

2 Electrophoresis enzyme BamHi digestion system in 1.5% agarose gel. But there is no band! We plan to do another PCR when the new primers come.

7-16

1 the new primer comes!

2 get the lpp-omp-mbp sequence with PCR. The PCR is carried out with two enzyme, the PFU and the Taq.

3 Electrophoresis PCR reaction system in 1.5% agarose gel. Now there is one and only one band of the PFU PCR system. The fragments is purified.

4 double digestion of both the carrier plasmid and the PCR fragments. The commercial plasmid PET21a and commercial fragments are digested with NdeI and XhoI. The standard plasmid with RBS and standard fragments are digested with XbaI and PstI.

5 ligation the lpp-ompa-mbp fragments to the vectors.

6 transform the ligation products.

7-17

pick the clones and shake to amplify the bactira.

7-18

1 miniprep of the lpp-ompa-mbp-PET21A and rbs-lpp-ompa-mbp-PSB1A2.

2 bacteria PCR is done to verify if the fragments have been ligated into the vector.

3 Electrophoresis PCR reaction system in 1.5% agarose gel. And I am very happy to see that the results are right!

4 the plasmid are sent to sequence for further valediction.

7-19

1 Double enzyme digestion of the rbs-lpp-ompa-mbp with XbaI and PstI.

2 Electrophoresis enzyme system in 1.5% agarose gel. And the productions of the enzyme digestion are purified.

3 ligation(rbs-lpp-ompa-mbp+T7promoter as vector)

4 transform both the lpp-ompa-mbp-PET21A to BL21 for test and the PT7-rbs-lpp-ompa-mbp to trans10.

7-20

1 the results of the sequence of the lpp-ompa-mbp-PET21a andrbs-lpp-ompa-mbp show that there are right clones.

2 amplify the PT7-rbs-lpp-ompa-mbp.

3 miniprep the plasmid and the plasmids are sent to sequence.

7-21

1 pick one positive clone of the lpp-ompa-mbp-PET21a(BL21) and shake in the 37℃.for10h

2 dilute 20ul in 5ml LB and shake the bacteria in 37℃ for 2~3h until the OD reaches to 0.6~0.8.

3 Add IPTG into the bacteria and shake in 30℃ for 4~5h.

4 send the bacteria to carry out Western blot.

7-23

1 The sequence results show that the number 1 and 4 PT7-rbs-lpp-ompa-mbp are right.

2 double digest the PT7-rbs-lpp-ompa-mbp(ES) and the terminator B0015(E X).

7-24

1 Electrophoresis enzyme system in 1.5% agarose gel. The results are right.

2 there was an accident when the gel is purified.

3 again double digest the PT7-rbs-lpp-ompa-mbp(ES) and the terminator B0015(E X).

7-25

1 Electrophoresis enzyme system in 1.5% agarose gel. The results are right and the gel is purified to get the fragments.

2 ligation(PT7-rbs-LOM+Terminator-PSB1AK3)

3 transformation the PT7-rbs-LOM to BL21 and PT7-rbs-LOM-Terminator-PSB1AK3 to trans5alpha.

4 pick the clones and bacteria PCR is done.

7-26

1 Electrophoresis PCR system in 1.5% agarose gel. The right plasmid are sent to sequence to verify.

2 induction the bacteria of BL21 with the plasmid lpp-ompa-mbp-PET21a and PT7-rbs-LOM to prepare for SDS-PAGE and Western blot.

7-27—7.31

1 induction the bacteria

2 carry out the experiment of SDS-PAGE.