Team:Peking/Notebook/DHLiang
From 2010.igem.org
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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== | ||
+ | {| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff" | ||
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+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.1|1]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.2|2]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.3-8.6|3]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.3-8.6|4]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.3-8.6|5]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.3-8.6|6]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.7|7]] | ||
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+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.8|8]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/DHLiang#8.9|9]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.10|10]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.11|11]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.12-8.14|12]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.12-8.14|13]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.12-8.14|14]] | ||
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+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.15-8.17|15]] | ||
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+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.15-8.17|17]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.18|18]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.19-8.21|19]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.19-8.21|20]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.19-8.21|21]] | ||
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+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.22-8.25|22]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.22-8.25|23]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/DHLiang#8.22-8.25|24]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.22-8.25|25]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.26|26]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.27-8.28|27]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.27-8.28|28]] | ||
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+ | |style="text-align:center"| [[Team:Peking/Notebook/DHLiang#8.29|29]] | ||
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+ | |} | ||
+ | [[https://2010.igem.org/Team:Peking/Notebook/DHLiang TOP]] |
Revision as of 15:42, 25 October 2010
Contents
July
Mon | Tue | Wed | Thu | Fri | Sat | Sun |
- | - | - | - | 1 | 2 | 3 |
4 | 5 | 6 | 7 | 8 | 9 | 10 |
11 | 12 | 13 | 14 | 15 | 16 | 17 |
18 | 19 | 20 | 21 | 22 | 23 | 24 |
25 | 26 | 27 | 28 | 29 | 30 | 31 |
[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
Mon | Tue | Wed | Thu | Fri | Sat | Sun |
1 | 2 | 3 | 4 | 5 | 6 | 7 |
8 | 9 | 10 | 11 | 12 | 13 | 14 |
15 | 16 | 17 | 18 | 19 | 20 | 21 |
22 | 23 | 24 | 25 | 26 | 27 | 28 |
29 | 30 | 31 | - | - | - | - |
[TOP]