Team:Peking/Notebook/DHLiang

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Contents

• July, 2010

• August, 2010

• September, 2010

• October, 2010

July

[TOP]

7.1

Dissolve primers

Design PCR programme

7.4

MerR PCR, MBP PCR

Retrieve the PCR product

7.5

Digest the plasmid pET-39(B)+ with SacII and EcoRI

Digest the PCR product with SacII and EcoRI

Retrieve the digested product

Ligated the digested plasmid pET-39(b)+ and the PCR product

Transform the ligation product into Trans5αstrain.

7.6

Part I handle the job for YHu

Digest pET-21a with NdeI and XhoI

Retrieve the digested product

Ligated the digested pET-21a with MBP digested fragments

Transform the ligation product

Part II Periplasmic Construction

Pick the six single clones for the plate transformed last night

PCR the clones to identify the successfully ligated clone

Clones 1\3\5 shows positive result and go on shaking at 37℃ overnight

7.7

Part I handle the job for YHu

Pick clones from the plate transformed yesterday and shake at 37℃ for ten hours

Miniprep the plasmid from the clones

Part II Periplasmic Construction

Miniprep clone 1\3\5 and send them for sequencing

7.8

Part I handle the job for YHu

Digest the plasmid minipreped yesterday and identify by Electrophoresis

No positive result showed

Redo the experiment starting with PCR the MBP

Part II Periplasmic Construction

Positive Transformation of DsbA-MerR

Start the construction of DsbA-MBP

PCR MBP overnight

7.9

Part I handle the job for YHu

Redo the ligation of MBP into pET-21a

Transformation

Part II Periplasmic Construction

The sequence of Clone 1 from DsbA-MerR is correct

Retrieve the MBP PCR product

7.10

YHu's job is officially handled by XTeng.I begin to conduct the experiment of the periplasmic module of the bioabsorbent display alone

Digest pET-39(b)+ and the retrieved MBP PCR product

Ligate the product overnight

Start the standardization of DsbA-MerR

PCR DsbA-MerR with standadized primers

7.11

Transformation of the ligated DsbA-MBP

Transformation of standardization of DsbA-MerR

7.12

Plasmid Miniprep from the transformation product

7.13

Digest the product of DsbA-MerR with EcoRI and SacII and do the identificate by Electrophoresis

Digest the product of standardization of DsbA-MerR with EcoRI and PstI and do the identificate by Electrophoresis

No postive result showed

7.14

re-Conduct the experiment

PCR MBP and retrieve the product

PCR DsbA-MerR and retrieve the product

Digest the product with EcoRI and SacII

Digest the the standardization product with EcoRI and PstI

Retrieve the product and ligate with digested pET-39(b)+

Transform the ligated product

7.15

Pick 24 clones from the plates and shake at 37℃ for ten hours

Start the construction of MerR into pET-21a

PCR MerR and retrieve

Digest it with XhoI and NdeI

Retrieve the digested product and ligate with digested pET-21a

7.16

Plasmid Miniprep from the 24 clones

Pick 21 clones from the plates of the standardization of DsbA-MerR and shake at 37℃ for ten hours

Digest the Plasmid Miniprep product and identificate by Electrophoresis

7.17

Positive result showed among the clones,they are A21 A22 A23 A27 C15 C21 C22

Positive transform of A22 A27 C15 C22

7.18-7.24

Go to ShangHai EXPO on a vocation.

The sequence result( done by Xteng) showed the construction of DsbA-MBP failed again.

Meanwhile since we later discovered there is a PstI restriction site right in the middle of DsbA ,unfortuanately we uesd to digest the Standardized PCR product of DsbA-MerR with PstI,so this part failed, too.

7.25

Digest pET-39(b)+ with XbaI and XhoI

PCR MBP and retrieve the product

Digest it with XbaI and XhoI

Ligate the digested vector and the PCR product

7.26

Transform the ligated product

Pick clones from the plate and shake at 37℃ ovenight

7.27

Plasmid Miniprep and send for sequencing

7.28

PCR DsbA-MerR

Positive transform DsbA-MerR into Omni Strain

7.29

The Sequence of DsbA-MBP result failed again, but reveal that the original plasmid containing MBP from Summers is incorrect.

We design to do the three fragment Ligation strategy to construct the MBP

PCR Strain NRI/PASK-MBD with two pairs of primers

Retrieve the product

Start the nested PCR of DsbA-MerR to finish its Standradization.

7.30

Digest the pEt-39(b)+ with XbaI and XhoI

Digest the MBD-Part I with XbaI and BamHi

Digest the MBD-Part II with XhoI and BamHi

Rerieve the three products and ligate them together for three hours

Transform the ligated product

Identificate the Standradization of DsbA-MerR but failed

7.31

Yesterday's transformation failed

Re-ligate the three fragments for three hours

Transform the product

Redo the first round nested PCR of DsbA-MerR

Retrieve the product

The Transformation seems to be successful and pick 12 clones from the plate

Do the second round nested PCR of DsbA-MerR

August

[TOP]

8.1

Retrieve the second round nested PCR product of DsbA-MerR

Identificate the No 442 444 332 (DsbA-MBP) by Electrophoresis

No Positive resluit in the Electrophoresis

Miniprep of the new redo construction of DsbA-MBP

8.2

Digest 442 444 332 product and check the reslut by Electrophoresis

No positive result again

Redo the PCR of MBP

8.3-8.6

Redo the construction of DsbA-MBP ( just as the steps on 7.30-8.1)

Standardization of DsbA-MerR was successfully constructed,confirmed by the sequencing result.

Start the western-blot of DsbA-MerR

8.7

Miniprep of 12 clones of DsbA-MBP

Identificate by Electrophoresis but no positive result

Send it for sequencing

8.8

Begin the construction of standardization of DsbA-MBP while it's waiting for sequencing

8.9

Redo the PCR of MBP into two fragments

Digest pET-39(b)+ again with Spe I and Xho I

Ligate the three fragments again

8.10

Transform the ligated product

Retrieve the PCR product of the three fragments

Digest with XbaI and BamHI for N and XhoI and BamHI for C

Retrieve the digested product and Ligate them with digested pET-39(b)+ together

Transform the ligated product

8.11

Pick clones from the plate and shake at 37℃ ovenight

8.12-8.14

Postive results showed some clones is correct

Send for sequencing

8.15-8.17

Sequencing result show that NIC1 6 clone is correct

DsbA-MBP construction is complete

Positive Transform DsbA-MBP

8.18

Transform the DsbA-MBP into Omni and BL21a

8.19-8.21

Begin the Standardization of DsbA-MBP

First Round of Nested PCR of the DsbA-MBP

Second Round of Nested PCR of the DsbA-MBP

Construction Finished and waited for sequence

8.22-8.25

Go home on vocation

8.26

Sequence result show that the standardization of DsbA-MBP is failed

8.27-8.28

Redo the Standardization of DsbA-MBP

Construction Finished and waited for sequence

8.29

Sequencing result correct

Start coworking with Ao Liu on the promoter characterization==September==