Team:Peking/Notebook/DHLiang

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Contents

• July, 2010

• August, 2010

• September, 2010

• October, 2010

July

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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==