Team:Peking/Notebook/YWChen

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Contents

• July, 2010

• August, 2010

• September, 2010

• October, 2010

July

7.26

PbrR MBP construction plan

August

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8.1

Ⅰ.Use Tag PCR "PbrR MBP(≈500bp)" for commercial plasmid(PET-21a) and standard plasmid (PSB1K3)

Ⅱ.Digestion:The products of PCR(PbrR MBP)[backbone 4100bp;digest site PstⅠ&NdeⅠ]

Ⅲ.Gel for identification & Retrieve the gel

Ⅳ.Miniprep pbrR-mbp-commercial for backups

8.2

Lpp-OmpA-MBP construction plan

Ⅰ.Use Pfu Mix PCR Nde1+Lpp-OmpA(437bp)+Sal1 & E(EcoR1)NX(Xba1)+Lpp-OmpA+Sal1

Ⅱ.Digest the PCR products by Ndel,Sall&EcoR,Sall ,respectively

Ⅲ.if the products above got right ,Retrieve the gel ,if not ,back to Ⅰuse gel retrieve kit then digestion for 2h,after that, go to purification.

8.3

MBP construction plan

Ⅰ.Pfu PCR Sall+N-MBP+Bspel

Bspel+C-MBP+SNP

Bspel+C-MBP+Xhol

Ⅱ.Identify them using agarose gel electrophoresis.if right ,go to retrieve the gel, if not ,back to Ⅰ

8.4

Lpp-OmpA,N-MBP,+C-MBP with backbone PET21a and PSBIK3

Ⅰ.Ligation,Nde1+Lpp-OmpA(437bp)+Sal1 with Sall+N-MBP+Bspel & Bspel+C-MBP+Xhol & PET21a

Ⅱ.Ligation, E(EcoR1)NX(Xba1)+Lpp-OmpA+Sal1 with Sall+N-MBP+Bspel & Bspel+C-MBP+SNP & PSBIK3

Ⅲ.Learn to do the western blotting. Write the protocols.

8.5

Transformation the products of 4 fragments above.

8.6

There is something wrong with the primer ,so it doesn't work, back to the work from 8.2

8.7

Ⅰ.Use Pfu Mix PCR Nde1+Lpp-OmpA(437bp)+Sal1 & E(EcoR1)NX(Xba1)+Lpp-OmpA+Sal1

Ⅱ.Digest the PCR products by Ndel,Sall&EcoR,Sall ,respectively

8.8

Ⅰ.Pfu PCR Sall+N-MBP+Bspel

Bspel+C-MBP+SNP

Bspel+C-MBP+Xhol

Ⅱ.Gel to identification

8.9

Ⅰ.Ligation,Nde1+Lpp-OmpA(437bp)+Sal1 with Sall+N-MBP+Bspel & Bspel+C-MBP+Xhol & PET21a

Ⅱ.Ligation, E(EcoR1)NX(Xba1)+Lpp-OmpA+Sal1 with Sall+N-MBP+Bspel & Bspel+C-MBP+SNP & PSBIK3

8.10

Transform the ligation product into Trans5α strain.

8.11

Help a teammate transform P(RBS+T3pol 1)&PmerR-PSB1C3 Plasmid to OmniMAX2-T1 Competent cell

Digestion:

Ⅰ.RBS+T3pol 1 with XbaI and pstI(insert)

Ⅱ.PmerR-PSB1C3 with SpeI and PstI (vector)

Retrieve the gel

Ligation the parts above.

8.12

Digestion MBP construct parts

Ligation 4 fragments

Nde1+Lpp-OmpA(437bp)+Sal1 with Sall+N-MBP+Bspel & Bspel+C-MBP+Xhol & PET21a//E(EcoR1)NX(Xba1)+Lpp-OmpA+Sal1 with Sall+N-MBP+Bspel & Bspel+C-MBP+SNP & PSBIK3 overnight

8.13

Digestion: PET21a 20 µl with NdeI and XhoI

Ligation again

Ⅰ.RBS+T3pol 1 with XbaI and pstI(insert)

Ⅱ.PmerR-PSB1C3 with SpeI and PstI (vector)

Insert:vector=1:7&2:6

Transform the ligation product

Finally got clone ,2:6 better than 1:7

Ⅲ.transformation 4 pieces fragments

8.14

Ⅰ.Plate PCR for identification 4 pieces fragments ligation products

Ⅱ.Miniprep

21a-(1~3) 1K3(1~3) 26-(1~2) 17-(3~4)

Ⅲ.Sent the plasmids for sequencing

8.15

Ⅰ.Got the right sequence 1K3(1~3) 26-(1~2) 17-(3~4) while 21a-(1~3) without MBP but have Lpp-OmpA inside

Ⅱ.transformation

1K3(1~3) 26-(1~2) 17-(3~4)

Ⅲ.Pick 20 single clones on 21a plate

Ⅳ.PCR for identification

Got the right size from agarose gel electrophoresis ( Lpp-OmpA+MBP=735bp)

Ⅴ.Digestion with NdeI and XhoI,then use PCR purification kit to retrieve products.

Ⅵ.Ligation 1(PET-21a):7

Ⅶ.Transformation

September

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9.5

Assembly:

T7p+RBS(B0034)+DsbA+MBP+TER(B0015)+T7p+RBS(B0034)+MBP+TER(B0015)+

T7p+RBS(B0034)+Lpp_OmpA+MBP+TER(B0015)

Ⅰ.Digestion:RBS with SpeI and PstI

1-23L terminator with EcoRI and XbaI

Ⅱ.Identify them using agarose gel electrophoresis.if right ,go to retrieve the gel

9.9

Ligation: RBS+MBP

DsbA+MBP

FORGET DIGESTION!!!!

9.10

Digestion:MBD with XbaI and PstI

DsbA-MBP with EcoRI and SpeI

Identify them using agarose gel electrophoresis.if right ,go to retrieve the gel

9.11

Transformation to BL21 for western blotting

Omni for preserve

9.13

Digestion :with XbaI and PstI (for ligation)

Miniprep T7p+RBS(B0034)+DsbA+MBP+Ter(1~3)&RBS+MBP（1~3）

9.15

Ⅰ.Sent the plasmids for sequencing【T7p+RBS(B0034)+DsbA+MBP+Ter(1~3)&RBS+MBP（1~3）】

T7p+RBS(B0034)+DsbA+MBP+Ter 1 got right sequence

Ⅱ.Digestion:

T7p+RBS(B0034)+DsbA+MBP+Ter with EcoRI and SpeI

Identify them using agarose gel electrophoresis.Then retrieve the gel(got the right size)

Ⅲ.Digestion:

RBS+MBP with XbaI and PstI

After Identify them by using agarose gel electrophoresis,the results turns wrong.

Digestion AGAIN!:

RBS+MBP with XbaI and PstI

Meanwhile: Pick 10 single clone for plate PCR by using tagmix

Digest the PCR products with XbaI and PstI ,10 clones all got the right size,so minipret them.

9.16

Ligation :RBS-MBP(XP)+Ter(SP)

Transformation it.

Culture the plates.

9.17

Pick the single clone to PCR, but failed

Meanwhile: Digest the RBS-MBP1 for backup

9.18

Digest the RBS-MBP(2&3)

Ligation: RBS-MBP(2&3) +T7p

Transformation the Ligation products

9.19

Use RBS-MBP(2&3) as templete,PCR(Fast pfu),got a mass of RBS-MBP

Identify them using agarose gel electrophoresis.Then go to retrieve the gel

9.20

Transformation:RBS-MBP-T7p

Culture the plates

9.21

Pick 3 single clone T7p-RBS-MBP(1~3)

Some for PCR some for Miniprep

9.22

Miniprep T7p-RBS-MBP(1~3)

Digestion:T7p-RBS-MBP(1~3) with EcoRI and SpeI

9.23

Wiki writing

Retrieve the digestion products T7p-RBS-MBP(1~3)ES

Ligation:

T7p-RBS-MBP(1~3)+Ter

Transformation:T7p-RBS-MBP-Ter(1~3)

Culture the plates

9.24

Pick 3 single clone from each plate(except1)

Miniprep T7p-RBS-MBP-Ter(2-1~3&3-1~3)

10µI for sequencing

10µI for digestion

Left 20µI

9.25

Digestion:T7p-RBS-MBP-Ter with SpeI and PstI

Gel for identification ,the size of T7p-RBS-MBP-Ter(2-1~3) are right

9.26

Digestion:PSB1C3 with EcoRI and PstI

Ligation:PSB1C3 + T7p-RBS-MBP-Ter+T7p+RBS+Lpp-OmpA+MBP+Ter

Gel retrieving

9.27

Transformation:Positive cloning

T7p+RBS+Lpp-OmpA+MBP+Ter&T7p+RBS(B0034)+DsbA+MBP+Ter

Culture the plates

9.28

The sequence of T7p-RBS-MBP-Ter(2-1~3&3-1~3) came out ,but without T7p inside.

October

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10.3

There is something wrong with PSBIC3(RFP inside )

Ligation Again!:

PSB1C3 + T7p-RBS-MBP-Ter+T7p+RBS+Lpp-OmpA+MBP+Ter

10.4

Pick 3 single clone from the plate

Miniprep them.

10.5

Transformation:Positive cloning:T7p+RBS(B0034)+DsbA+MBP+Ter

Digestion:RBS-MBP-Ter+T7p with XbaI and PstI

Identify them using agarose gel electrophoresis.Then go to retrieve the gel

10.6

Pick 3 single clone from the plate（T7p+RBS(B0034)+DsbA+MBP+Ter）

Digestion:PSB1C3 with XbaI and PstI （Change backbone）

Identify them using agarose gel electrophoresis.Then go to retrieve the gel

10.7

Miniprep T7p+RBS(B0034)+DsbA+MBP+Ter(1~3)

Digestion：RBS-MBP-Ter with XbaI and PstI

Ligation:RBS-MBP-Ter+T7p(sp) then transformation

10.8

Ligation again

T7p+RBS-MBP-Ter-+T7p+RBS+Lpp-OmpA+MBP+Ter

Transformation it

10.9

Transformation T7p+RBS+Lpp-OmpA+MBP+Ter(xp)-PSB1C3(xp)

PCR(tag mix) for identification T7p+RBS(B0034)+DsbA+MBP+Ter

Culture the plates, but it did not grow up

10.10

Ligation:T7p+RBS+Lpp-OmpA+MBP+Ter(EP)+PSB1C3

T7p+RBS+DsbA+MBP+Ter(EP)+PSB1C3

Transformation

Easy Mix PCR.

Culture the plates

10.11

ⅠDigestion:T7p+RBS+Lpp-OmpA+MBP+Ter(EP)

T7p+RBS+DsbA+MBP+Ter(EP)

Identify them using agarose gel electrophoresis.Then go to retrieve the gel

10.16

Ⅰ.Ligation: T7p-RBS-MBP-Ter

Ⅱ.Transformation :T7p-RBS-MBP-Ter

Ⅲ.Culture the plates

Pick 3 single clones

10.17

T7p-RBS-MBP-Ter for sequencing

10.20

Ⅰ.Liagation:T7p+RBS+DsbA+MBP+TER+T7p+RBS+MBP+TER+T7p+RBS+Lpp-OmpA+MBP+TER+PSBIC3

Ⅱ.Transformation

Ⅲ.Culture the plates

Pick 6 Single clones

Ⅳ.T7p+RBS+DsbA+MBP+TER+T7p+RBS+MBP+TER+T7p+RBS+Lpp-OmpA+MBP+TER+PSBIC3 for sequencing

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