Team:Peking/Notebook/DHLiang

From 2010.igem.org

(Difference between revisions)
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===7.1===
===7.1===
Dissolve primers
Dissolve primers
 +
Design PCR programme
Design PCR programme
===7.4===
===7.4===
MerR PCR, MBP PCR
MerR PCR, MBP PCR
 +
Retrieve the PCR product
Retrieve the PCR product
===7.5===
===7.5===
Digest the plasmid pET-39(B)+ with SacII and EcoRI
Digest the plasmid pET-39(B)+ with SacII and EcoRI
 +
Digest the PCR product with SacII and EcoRI
Digest the PCR product with SacII and EcoRI
 +
Retrieve the digested product
Retrieve the digested product
 +
Ligated the digested plasmid pET-39(b)+ and the PCR product
Ligated the digested plasmid pET-39(b)+ and the PCR product
 +
Transform the ligation product into Trans5αstrain.
Transform the ligation product into Trans5αstrain.
===7.6===
===7.6===
Part I handle the job for YHu
Part I handle the job for YHu
 +
Digest pET-21a with NdeI and XhoI
Digest pET-21a with NdeI and XhoI
 +
Retrieve the digested product
Retrieve the digested product
 +
Ligated the digested pET-21a with MBP digested fragments
Ligated the digested pET-21a with MBP digested fragments
 +
Transform the ligation product
Transform the ligation product
 +
Part II  Periplasmic Construction
Part II  Periplasmic Construction
 +
Pick the six single clones for the plate transformed last night
Pick the six single clones for the plate transformed last night
 +
PCR the clones to identify the successfully ligated clone
PCR the clones to identify the successfully ligated clone
 +
Clones 1\3\5 shows positive result and go on shaking at 37℃ overnight
Clones 1\3\5 shows positive result and go on shaking at 37℃ overnight
===7.7===
===7.7===
Part I handle the job for YHu
Part I handle the job for YHu
 +
Pick clones from the plate transformed yesterday and shake at 37℃ for ten hours
Pick clones from the plate transformed yesterday and shake at 37℃ for ten hours
 +
Miniprep the plasmid from the clones
Miniprep the plasmid from the clones
 +
Part II  Periplasmic Construction
Part II  Periplasmic Construction
 +
Miniprep clone 1\3\5 and send them for sequencing
Miniprep clone 1\3\5 and send them for sequencing
===7.8===
===7.8===
Part I handle the job for YHu
Part I handle the job for YHu
 +
Digest the plasmid minipreped yesterday and identify by Electrophoresis
Digest the plasmid minipreped yesterday and identify by Electrophoresis
 +
No positive result showed
No positive result showed
 +
Redo the experiment starting with PCR the MBP
Redo the experiment starting with PCR the MBP
 +
Part II  Periplasmic Construction
Part II  Periplasmic Construction
 +
Positive Transformation of DsbA-MerR
Positive Transformation of DsbA-MerR
 +
Start the construction of DsbA-MBP
Start the construction of DsbA-MBP
 +
PCR MBP overnight
PCR MBP overnight
===7.9===
===7.9===
Part I handle the job for YHu
Part I handle the job for YHu
 +
Redo the ligation of MBP into pET-21a
Redo the ligation of MBP into pET-21a
 +
Transformation
Transformation
 +
Part II  Periplasmic Construction
Part II  Periplasmic Construction
 +
The sequence of Clone 1 from DsbA-MerR is correct
The sequence of Clone 1 from DsbA-MerR is correct
 +
Retrieve the MBP PCR product
Retrieve the MBP PCR product
===7.10===
===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
+
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
Digest pET-39(b)+ and the retrieved MBP PCR product
 +
Ligate the product overnight  
Ligate the product overnight  
 +
Start the standardization of DsbA-MerR
Start the standardization of DsbA-MerR
 +
PCR DsbA-MerR with standadized primers
PCR DsbA-MerR with standadized primers
===7.11===
===7.11===
Transformation of the ligated DsbA-MBP
Transformation of the ligated DsbA-MBP
 +
Transformation of standardization of DsbA-MerR
Transformation of standardization of DsbA-MerR
===7.12===
===7.12===
Line 157: Line 193:
===7.13===  
===7.13===  
Digest the product of DsbA-MerR with EcoRI and SacII and do the identificate by Electrophoresis
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
Digest the product of standardization of DsbA-MerR with EcoRI and PstI and do the identificate by Electrophoresis
 +
No postive result showed
No postive result showed
===7.14===
===7.14===
re-Conduct the experiment
re-Conduct the experiment
 +
PCR MBP and retrieve the product
PCR MBP and retrieve the product
 +
PCR DsbA-MerR and retrieve the product
PCR DsbA-MerR and retrieve the product
 +
Digest the product with EcoRI and SacII
Digest the product with EcoRI and SacII
 +
Digest the the standardization product with EcoRI and PstI
Digest the the standardization product with EcoRI and PstI
 +
Retrieve the product and ligate with digested pET-39(b)+
Retrieve the product and ligate with digested pET-39(b)+
 +
Transform the ligated product
Transform the ligated product
===7.15===
===7.15===
Pick 24 clones from the plates and shake at 37℃ for ten hours
Pick 24 clones from the plates and shake at 37℃ for ten hours
 +
Start the construction of MerR into pET-21a
Start the construction of MerR into pET-21a
 +
PCR MerR and retrieve
PCR MerR and retrieve
 +
Digest it with XhoI and NdeI  
Digest it with XhoI and NdeI  
 +
Retrieve the digested product and ligate with digested pET-21a
Retrieve the digested product and ligate with digested pET-21a
===7.16===
===7.16===
Plasmid Miniprep from the 24 clones
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
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
Digest the Plasmid Miniprep product and identificate by Electrophoresis
===7.17===
===7.17===
Positive result showed among the clones,they are A21 A22 A23 A27 C15 C21 C22
Positive result showed among the clones,they are A21 A22 A23 A27 C15 C21 C22
 +
Positive transform of A22 A27 C15 C22
Positive transform of A22 A27 C15 C22
===7.18-7.24===
===7.18-7.24===
Go to ShangHai EXPO on a vocation.
Go to ShangHai EXPO on a vocation.
 +
The sequence result( done by Xteng) showed the construction of DsbA-MBP failed again.
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.
+
 
 +
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===
===7.25===
Digest pET-39(b)+ with XbaI and XhoI
Digest pET-39(b)+ with XbaI and XhoI
 +
PCR MBP and retrieve the product
PCR MBP and retrieve the product
 +
Digest it with XbaI and XhoI
Digest it with XbaI and XhoI
 +
Ligate the digested vector and the PCR product
Ligate the digested vector and the PCR product
===7.26===
===7.26===
Transform the ligated product
Transform the ligated product
 +
Pick clones from the plate and shake at 37℃ ovenight
Pick clones from the plate and shake at 37℃ ovenight
===7.27===
===7.27===
Line 196: Line 254:
===7.28===
===7.28===
PCR DsbA-MerR
PCR DsbA-MerR
 +
Positive transform DsbA-MerR into Omni Strain
Positive transform DsbA-MerR into Omni Strain
===7.29===
===7.29===
-
The Sequence of DsbA-MBP result failed again, but reveal that the original plasmid containing MBP from Summers is incorrect.
+
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
We design to do the three fragment Ligation strategy to construct the MBP
 +
PCR Strain NRI/PASK-MBD with two pairs of primers
PCR Strain NRI/PASK-MBD with two pairs of primers
 +
Retrieve the product
Retrieve the product
 +
Start the nested PCR of DsbA-MerR to finish its Standradization.
Start the nested PCR of DsbA-MerR to finish its Standradization.
===7.30===
===7.30===
Digest the pEt-39(b)+ with XbaI and XhoI
Digest the pEt-39(b)+ with XbaI and XhoI
 +
Digest the MBD-Part I with XbaI and BamHi
Digest the MBD-Part I with XbaI and BamHi
 +
Digest the MBD-Part II with XhoI and BamHi
Digest the MBD-Part II with XhoI and BamHi
 +
Rerieve the three products and ligate them together for three hours
Rerieve the three products and ligate them together for three hours
 +
Transform the ligated product
Transform the ligated product
 +
Identificate the Standradization of DsbA-MerR but failed
Identificate the Standradization of DsbA-MerR but failed
===7.31===
===7.31===
Yesterday's transformation failed
Yesterday's transformation failed
 +
Re-ligate the three fragments for three hours
Re-ligate the three fragments for three hours
 +
Transform the product
Transform the product
-
redo the first round nested PCR of DsbA-MerR
+
 
 +
Redo the first round nested PCR of DsbA-MerR
 +
 
Retrieve the product
Retrieve the product
-
the Transformation seems to be successful and pick 12 clones from the plate
+
 
 +
The Transformation seems to be successful and pick 12 clones from the plate
 +
 
Do the second round nested PCR of DsbA-MerR==August==
Do the second round nested PCR of DsbA-MerR==August==

Revision as of 15:35, 25 October 2010





   Donghai Liang's Notes
                                                                                                                                                goto his page
Being responsible for the periplasmic module of the bioabsorbent display, I successfully completed the construction of DsbA-MBP and DsbA-MerR (serving as a control). At the same time, the standardization of the module mentioned above is also completed. Besides, I participate in the TF-DNA binding affinity characterization of the mercury promoter

download his notes

Contents


July

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[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==

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