Team:Peking/Notebook/MJing

From 2010.igem.org

(Difference between revisions)
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===7.3===
===7.3===
Antigen 43 PCR, 20ul system. T3 polymerase PCR.
Antigen 43 PCR, 20ul system. T3 polymerase PCR.
 +
Identify them by 1% agarose gel electrophoresis.
Identify them by 1% agarose gel electrophoresis.
===7.4===
===7.4===
Antigen 43 PCR using different annealing temperature, with gradient 1 ℃.  
Antigen 43 PCR using different annealing temperature, with gradient 1 ℃.  
 +
Attend the group seminar.
Attend the group seminar.
 +
Retrieve the PCR product of T3 polymerase and digest it with EcoRI and PstI. Digest for the whole night.
Retrieve the PCR product of T3 polymerase and digest it with EcoRI and PstI. Digest for the whole night.
===7.5===
===7.5===
Identify the product by agarose gel electrophoresis. Retrieve the PCR product of Antigen 43.
Identify the product by agarose gel electrophoresis. Retrieve the PCR product of Antigen 43.
 +
Retrieve the digested T3 polymerase and ligated it to the plasmid PSB1A2.
Retrieve the digested T3 polymerase and ligated it to the plasmid PSB1A2.
 +
Transform the plasmid to Trans 5α strain.
Transform the plasmid to Trans 5α strain.
 +
PCR the antigen 43 with much larger annealing temperature gradient. Identify it with electrophoresis.  
PCR the antigen 43 with much larger annealing temperature gradient. Identify it with electrophoresis.  
===7.6===
===7.6===
Line 114: Line 120:
===7.8===
===7.8===
PCR antigen 43 using nest-PCR procedure. Using Taq DNA polymerase to do the first step nest PCR.
PCR antigen 43 using nest-PCR procedure. Using Taq DNA polymerase to do the first step nest PCR.
-
Identify it by electrophoresis. Retrieve the 3k band and do the second step of nest PCR by using the product of the first step as template.
+
 
 +
Identify it by electrophoresis. Retrieve the 3k band and do the second step of nest PCR by using the product of the  
 +
first step as template.
===7.9===
===7.9===
-
Identify the second PCR product by electrophoresis. Retrieve the 3k band and digest it with EcoRI and SpeI to put it into plasmid. Digest it with PstI to test whether this is antigen 43.( antigen 43 have 6 PstI digestion sites.)
+
Identify the second PCR product by electrophoresis. Retrieve the 3k band and digest it with EcoRI and SpeI to put it  
 +
into plasmid. Digest it with PstI to test whether this is antigen 43.( antigen 43 have 6 PstI digestion sites.)
 +
 
Design the six point mutation primer.
Design the six point mutation primer.
===7.10===
===7.10===
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===7.12===
===7.12===
Prepare plasmid DNA.  
Prepare plasmid DNA.  
 +
Digest the plasmid DNA with PstI to identify it.  
Digest the plasmid DNA with PstI to identify it.  
 +
PCR the product using Easytaq DNA polymerase to identify the molecule weight of the product.
PCR the product using Easytaq DNA polymerase to identify the molecule weight of the product.
===7.13===
===7.13===
Learn to do the western blotting. Write the protocols.
Learn to do the western blotting. Write the protocols.
 +
Digest merT, merP and merC with Xbal and PstI. Meanwhile digest B0034(RBS) with SpeI and PstI.
Digest merT, merP and merC with Xbal and PstI. Meanwhile digest B0034(RBS) with SpeI and PstI.
 +
Connect the two digested product together.
Connect the two digested product together.
 +
Transform the ligation product into Trans5αstrain.
Transform the ligation product into Trans5αstrain.
 +
Pick the single clone from the plate.
Pick the single clone from the plate.
 +
Send the PCR product of Antigen 43 for sequencing.
Send the PCR product of Antigen 43 for sequencing.
===7.14===
===7.14===
Prepare the plasmid DNA of rbs+merT, rbs+merP, rbs+merC.
Prepare the plasmid DNA of rbs+merT, rbs+merP, rbs+merC.
 +
Do the western blotting.
Do the western blotting.
===7.15===
===7.15===
Digest the plasmid DNA by EcoRI and PstI to identify it.
Digest the plasmid DNA by EcoRI and PstI to identify it.
 +
Digest rbs+merT with Spel and Pstl and digest rbs+merP with Xbal and Pstl.
Digest rbs+merT with Spel and Pstl and digest rbs+merP with Xbal and Pstl.
 +
Identify them using agarose gel electrophoresis.
Identify them using agarose gel electrophoresis.
===7.16===
===7.16===
Connect rbs with merT, merP and merC again.  
Connect rbs with merT, merP and merC again.  
-
Do the first point mutation. First PCR the antigen 43 gene with designed point mutation primers, then retrieve it by electrophoresis. Then do the blunting kination, finally do the ligation and put it into strains by transformation.
+
 
 +
Do the first point mutation. First PCR the antigen 43 gene with designed point mutation primers, then retrieve it by  
 +
electrophoresis. Then do the blunting kination, finally do the ligation and put it into strains by transformation.
===7.18===
===7.18===
Prepare the plasmid DNA for rbs+merT, rbs+merP and rbs+merC, Antigen 43 mutant 1.  
Prepare the plasmid DNA for rbs+merT, rbs+merP and rbs+merC, Antigen 43 mutant 1.  
 +
Identify them using electrophoresis.
Identify them using electrophoresis.
 +
Connect rbs+merT with rbs+merP.
Connect rbs+merT with rbs+merP.
 +
Do the second step of point mutation for antigen 43. Do the PCR step. Identify it by electrophoresis.
Do the second step of point mutation for antigen 43. Do the PCR step. Identify it by electrophoresis.
Blunting kination, ligation and transformation.
Blunting kination, ligation and transformation.
 +
Send the first point mutation product for sequencing.
Send the first point mutation product for sequencing.
 +
The rbs+merT,rbs+merP and rbs+merC sequenced correct.
The rbs+merT,rbs+merP and rbs+merC sequenced correct.
===7.19===
===7.19===
Retrieve the product of digested rbs+merT and rbs+merP by electrophoresis. Connect the rbs+merT with rbs+merP,. Do the transformation.  
Retrieve the product of digested rbs+merT and rbs+merP by electrophoresis. Connect the rbs+merT with rbs+merP,. Do the transformation.  
 +
Pick the single clone of antigen 43 second step point mutation strain.
Pick the single clone of antigen 43 second step point mutation strain.
===7.20===
===7.20===
Prepare the plasmid DNA of antigen 43 second point mutation strain. Send it for sequencing.
Prepare the plasmid DNA of antigen 43 second point mutation strain. Send it for sequencing.
 +
Do the third step point mutation.  
Do the third step point mutation.  
 +
Identify the plasmid DNA by electrophoresis. Retrieve the third point mutation PCR product by electrophoresis.
Identify the plasmid DNA by electrophoresis. Retrieve the third point mutation PCR product by electrophoresis.
 +
Digest rbs+merT+rbs+merP with Spel and Pstl, digest rbs+merC with Xbal and Pstl.  
Digest rbs+merT+rbs+merP with Spel and Pstl, digest rbs+merC with Xbal and Pstl.  
===7.21===
===7.21===
Identify the product of the digestion using electrophoresis.  
Identify the product of the digestion using electrophoresis.  
 +
Pick the single clone of the 3rd point mutation of antigen 43 strain on the plate.
Pick the single clone of the 3rd point mutation of antigen 43 strain on the plate.
===7.22===
===7.22===
Connect rbs+merT+rbs+merP with rbs+merC.  
Connect rbs+merT+rbs+merP with rbs+merC.  
 +
Using easyPFu to PCR antigen 43.The forward primer contains a rbs. Digest it with EcoRl and Spel to put it into PSB1A2 plasmid.
Using easyPFu to PCR antigen 43.The forward primer contains a rbs. Digest it with EcoRl and Spel to put it into PSB1A2 plasmid.
 +
Prepare the plasmid DNA of the product of the third point mutation of antigen 43.digest it with EcoRl and SpeI to identify it.
Prepare the plasmid DNA of the product of the third point mutation of antigen 43.digest it with EcoRl and SpeI to identify it.
 +
Do the 4th point mutation PCR step.
Do the 4th point mutation PCR step.
===7.23===
===7.23===
Identify the plasmid using agarose gel electrophoresis.
Identify the plasmid using agarose gel electrophoresis.
 +
Send the plasmid DNA for sequencing.
Send the plasmid DNA for sequencing.
 +
Put the rbs+agn43 into plasmid by ligation.
Put the rbs+agn43 into plasmid by ligation.
===7.29===
===7.29===
Attend the group seminar.
Attend the group seminar.
 +
Prepare the plasmid DNA for rbs+merT+rbs+merP+rbs+merC. Digest it with EcoRl and Pstl for identification. Digest it with Xbal and Pstl and digest the plasmid contains T7 promoter with Spel and Pstl.
Prepare the plasmid DNA for rbs+merT+rbs+merP+rbs+merC. Digest it with EcoRl and Pstl for identification. Digest it with Xbal and Pstl and digest the plasmid contains T7 promoter with Spel and Pstl.
 +
Do the 5th point mutation of antigen 43. Do the PCR step.
Do the 5th point mutation of antigen 43. Do the PCR step.
 +
Identify the product of the 4th point mutation and the rbs+merT+rbs+merP+rbs+merC by electrophoresis. Collect those correct ones and send them for sequencing.
Identify the product of the 4th point mutation and the rbs+merT+rbs+merP+rbs+merC by electrophoresis. Collect those correct ones and send them for sequencing.
 +
See the point mutation result in the sequence.
See the point mutation result in the sequence.
 +
Identify the T7+rbs+merT+rbs+merP+rbs+merC by electrophoresis.
Identify the T7+rbs+merT+rbs+merP+rbs+merC by electrophoresis.
===7.30===
===7.30===
Digest rbs+agn43 with EcoRl and Xbal. Digest phiR73+Po promoter with EcoRl and Spel.
Digest rbs+agn43 with EcoRl and Xbal. Digest phiR73+Po promoter with EcoRl and Spel.
 +
Retrieve the digested T7+rbs+merT+rbs+merP+rbs+merC gel.  
Retrieve the digested T7+rbs+merT+rbs+merP+rbs+merC gel.  
 +
Do the transformation.
Do the transformation.
===7.31===
===7.31===
Digest 2-2E constitutive promoter with EcoRl and Spel. Digest rbs+agn43 with EcoRl and Xbal.
Digest 2-2E constitutive promoter with EcoRl and Spel. Digest rbs+agn43 with EcoRl and Xbal.
 +
Connect 2-2E promoter with rbs+agn43.
Connect 2-2E promoter with rbs+agn43.

Revision as of 10:55, 25 October 2010




   Jing Miao's Notes
                                                                                                                                                goto his page
I designed to engineer this operon under the regulation of T7 promoter, which is regulated by upstream T7 polymerase, to fulfill the goal that the absorbance of Hg(II) will be enhanced for an efficient bioabsorbent. Also I cloned and assembled agn43 into the inductive aggregation module. Agn43 is drove by PO promoter, which is the terminal of a cascade amplification


download her notes

Contents


July

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[TOP]

7.3

Antigen 43 PCR, 20ul system. T3 polymerase PCR.

Identify them by 1% agarose gel electrophoresis.

7.4

Antigen 43 PCR using different annealing temperature, with gradient 1 ℃.

Attend the group seminar.

Retrieve the PCR product of T3 polymerase and digest it with EcoRI and PstI. Digest for the whole night.

7.5

Identify the product by agarose gel electrophoresis. Retrieve the PCR product of Antigen 43.

Retrieve the digested T3 polymerase and ligated it to the plasmid PSB1A2.

Transform the plasmid to Trans 5α strain.

PCR the antigen 43 with much larger annealing temperature gradient. Identify it with electrophoresis.

7.6

Design the primer of antigen 43 again by Primer Premier 5.0.

7.8

PCR antigen 43 using nest-PCR procedure. Using Taq DNA polymerase to do the first step nest PCR.

Identify it by electrophoresis. Retrieve the 3k band and do the second step of nest PCR by using the product of the first step as template.

7.9

Identify the second PCR product by electrophoresis. Retrieve the 3k band and digest it with EcoRI and SpeI to put it into plasmid. Digest it with PstI to test whether this is antigen 43.( antigen 43 have 6 PstI digestion sites.)

Design the six point mutation primer.

7.10

Do the transformation and ligation.

7.12

Prepare plasmid DNA.

Digest the plasmid DNA with PstI to identify it.

PCR the product using Easytaq DNA polymerase to identify the molecule weight of the product.

7.13

Learn to do the western blotting. Write the protocols.

Digest merT, merP and merC with Xbal and PstI. Meanwhile digest B0034(RBS) with SpeI and PstI.

Connect the two digested product together.

Transform the ligation product into Trans5αstrain.

Pick the single clone from the plate.

Send the PCR product of Antigen 43 for sequencing.

7.14

Prepare the plasmid DNA of rbs+merT, rbs+merP, rbs+merC.

Do the western blotting.

7.15

Digest the plasmid DNA by EcoRI and PstI to identify it.

Digest rbs+merT with Spel and Pstl and digest rbs+merP with Xbal and Pstl.

Identify them using agarose gel electrophoresis.

7.16

Connect rbs with merT, merP and merC again.

Do the first point mutation. First PCR the antigen 43 gene with designed point mutation primers, then retrieve it by electrophoresis. Then do the blunting kination, finally do the ligation and put it into strains by transformation.

7.18

Prepare the plasmid DNA for rbs+merT, rbs+merP and rbs+merC, Antigen 43 mutant 1.

Identify them using electrophoresis.

Connect rbs+merT with rbs+merP.

Do the second step of point mutation for antigen 43. Do the PCR step. Identify it by electrophoresis. Blunting kination, ligation and transformation.

Send the first point mutation product for sequencing.

The rbs+merT,rbs+merP and rbs+merC sequenced correct.

7.19

Retrieve the product of digested rbs+merT and rbs+merP by electrophoresis. Connect the rbs+merT with rbs+merP,. Do the transformation.

Pick the single clone of antigen 43 second step point mutation strain.

7.20

Prepare the plasmid DNA of antigen 43 second point mutation strain. Send it for sequencing.

Do the third step point mutation.

Identify the plasmid DNA by electrophoresis. Retrieve the third point mutation PCR product by electrophoresis.

Digest rbs+merT+rbs+merP with Spel and Pstl, digest rbs+merC with Xbal and Pstl.

7.21

Identify the product of the digestion using electrophoresis.

Pick the single clone of the 3rd point mutation of antigen 43 strain on the plate.

7.22

Connect rbs+merT+rbs+merP with rbs+merC.

Using easyPFu to PCR antigen 43.The forward primer contains a rbs. Digest it with EcoRl and Spel to put it into PSB1A2 plasmid.

Prepare the plasmid DNA of the product of the third point mutation of antigen 43.digest it with EcoRl and SpeI to identify it.

Do the 4th point mutation PCR step.

7.23

Identify the plasmid using agarose gel electrophoresis.

Send the plasmid DNA for sequencing.

Put the rbs+agn43 into plasmid by ligation.

7.29

Attend the group seminar.

Prepare the plasmid DNA for rbs+merT+rbs+merP+rbs+merC. Digest it with EcoRl and Pstl for identification. Digest it with Xbal and Pstl and digest the plasmid contains T7 promoter with Spel and Pstl.

Do the 5th point mutation of antigen 43. Do the PCR step.

Identify the product of the 4th point mutation and the rbs+merT+rbs+merP+rbs+merC by electrophoresis. Collect those correct ones and send them for sequencing.

See the point mutation result in the sequence.

Identify the T7+rbs+merT+rbs+merP+rbs+merC by electrophoresis.

7.30

Digest rbs+agn43 with EcoRl and Xbal. Digest phiR73+Po promoter with EcoRl and Spel.

Retrieve the digested T7+rbs+merT+rbs+merP+rbs+merC gel.

Do the transformation.

7.31

Digest 2-2E constitutive promoter with EcoRl and Spel. Digest rbs+agn43 with EcoRl and Xbal.

Connect 2-2E promoter with rbs+agn43.

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