Team:Peking/Notebook/MJing

From 2010.igem.org

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[[https://2010.igem.org/Team:Peking/Notebook/MJing TOP]]
[[https://2010.igem.org/Team:Peking/Notebook/MJing TOP]]
-
===7.1===
+
===7.3===
-
 
+
Antigen 43 PCR, 20ul system. T3 polymerase PCR.
-
1. DNA double digestion of mbp fragments for 3 hours: NdeI/XhoI for commercialization and XbaI/PstI for standardization.  
+
Identify them by 1% agarose gel electrophoresis.
-
 
+
===7.4===
-
20uL reaction system:   
+
Antigen 43 PCR using different annealing temperature, with gradient 1 ℃.
-
 
+
Attend the group seminar.
-
Buffer4    2uL 
+
Retrieve the PCR product of T3 polymerase and digest it with EcoRI and PstI. Digest for the whole night.
-
 
+
===7.5===
-
NdeI      1.5uL
+
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.
-
XhoI      1.5uL
+
Transform the plasmid to Trans 5α strain.
-
 
+
PCR the antigen 43 with much larger annealing temperature gradient. Identify it with electrophoresis.
-
mbp(com)  15uL
+
===7.6===
-
 
+
Design the primer of antigen 43 again by Primer Premier 5.0.
-
 
+
===7.8===
-
Buffer3    2uL
+
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.
-
XbaI      1.5uL
+
===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.)
-
PstI     1.5uL
+
Design the six point mutation primer.
-
 
+
===7.10===
-
mbp(SD)   15uL
+
Do the transformation and ligation.
-
 
+
===7.12===
-
2. DNA Purification.
+
Prepare plasmid DNA.  
-
 
+
Digest the plasmid DNA with PstI to identify it.
-
===7.17===
+
PCR the product using Easytaq DNA polymerase to identify the molecule weight of the product.
-
1. PCR reaction for mbp fragments using dissolved primers.  
+
===7.13===
-
 
+
Learn to do the western blotting. Write the protocols.
-
Primers Reverse is the new ones with strepp tag.
+
Digest merT, merP and merC with Xbal and PstI. Meanwhile digest B0034(RBS) with SpeI and PstI.
-
 
+
Connect the two digested product together.
-
10x easypfu buffer    2uL
+
Transform the ligation product into Trans5αstrain.
-
 
+
Pick the single clone from the plate.
-
ddNTP Mixture        2uL
+
Send the PCR product of Antigen 43 for sequencing.
-
 
+
===7.14===
-
For                1uL
+
Prepare the plasmid DNA of rbs+merT, rbs+merP, rbs+merC.
-
 
+
Do the western blotting.
-
Rev                1uL
+
===7.15===
-
 
+
Digest the plasmid DNA by EcoRI and PstI to identify it.
-
Template plasmid     0.5uL
+
Digest rbs+merT with Spel and Pstl and digest rbs+merP with Xbal and Pstl.
-
 
+
Identify them using agarose gel electrophoresis.
-
phusionHF            0.2uL
+
===7.16===
-
 
+
Connect rbs with merT, merP and merC again.  
-
ddH2O              up to 20 uL
+
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.
-
 
+
-
2. Electrophoresis PCR reaction system in 1% agarose gel.
+
-
 
+
-
3. Excise the gel slice and extract the mbp fragments.
+
===7.18===
===7.18===
-
1. DNA double digestion of mbp fragments and merr plasmids.
+
Prepare the plasmid DNA for rbs+merT, rbs+merP and rbs+merC, Antigen 43 mutant 1.  
-
 
+
Identify them using electrophoresis.
-
2. ligation of insert DNA fragments into plasmid vector DNA for 1 hour
+
Connect rbs+merT with rbs+merP.
-
 
+
Do the second step of point mutation for antigen 43. Do the PCR step. Identify it by electrophoresis.
-
mbp(NdeI/XhoI)    6uL
+
Blunting kination, ligation and transformation.
-
 
+
Send the first point mutation product for sequencing.
-
pET-21a            2uL
+
The rbs+merT,rbs+merP and rbs+merC sequenced correct.
-
 
+
-
10x ligation buffer    1uL
+
-
 
+
-
T4 DNA Ligase      1uL
+
-
 
+
-
 
+
-
mbp (Xbal/PstI)          6uL
+
-
 
+
-
B0034 (rbs)              2uL
+
-
 
+
-
10x ligation buffer        1uL
+
-
 
+
-
T4 DNA Ligase            1uL
+
-
 
+
-
 
+
-
merr (Xbal/PstI)          6uL
+
-
 
+
-
B0034 (rbs)              2uL
+
-
 
+
-
10x ligation buffer        1uL
+
-
 
+
-
T4 DNA Ligase            1uL
+
-
 
+
-
3. Transformation
+
-
 
+
===7.19===
===7.19===
-
1. Miniprep of rbs-merr
+
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.
-
2. Digestion and identification by Electrophoresis
+
-
 
+
-
Buffer3    1uL 
+
-
 
+
-
XbaI      0.75uL
+
-
 
+
-
PstI      0.75uL
+
-
 
+
-
rbs-merr  7.5uL
+
-
 
+
===7.20===
===7.20===
-
Miniprep of 12 colonies of mbp-commercialization.
+
Prepare the plasmid DNA of antigen 43 second point mutation strain. Send it for sequencing.
-
 
+
Do the third step point mutation.
-
No colonies formation of rbs-mbp.
+
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===
===7.21===
-
1. DNA double digestion of colonies of mbp-pET-21a and rbs-merr.
+
Identify the product of the digestion using electrophoresis.  
-
 
+
Pick the single clone of the 3rd point mutation of antigen 43 strain on the plate.
-
10uL reaction system:   
+
-
 
+
-
Buffer4      1uL 
+
-
 
+
-
NdeI        0.75uL
+
-
 
+
-
XhoI          0.75uL
+
-
 
+
-
mbp-pET-21a  7.5uL
+
-
 
+
-
 
+
-
Buffer3    1uL 
+
-
 
+
-
XbaI      0.75uL
+
-
 
+
-
PstI      0.75uL
+
-
 
+
-
rbs-mbp  7.5uL
+
-
 
+
-
2. Electrophoresis in 1% agarose gel.
+
-
 
+
-
Sequence mbp_ commercialization
+
-
 
+
-
(The result is confirmed to be incorrect.)
+
-
 
+
-
3. ligation of rbs-mbp-1 and 2 into plasmid vector T7 for 1 hour
+
-
 
+
-
rbs-mbp(Xbal/PstI)    6uL
+
-
 
+
-
T7 vector(SpeI/PstI)    2uL
+
-
 
+
-
10x ligation buffer      1uL
+
-
 
+
-
T4 DNA Ligase        1uL
+
-
 
+
-
4. Transformation
+
-
 
+
===7.22===
===7.22===
-
No colonies formation of T7-rbs-merr. Do ligation and transformation again.
+
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.
-
===7.23===
+
Prepare the plasmid DNA of the product of the third point mutation of antigen 43.digest it with EcoRl and SpeI to identify it.
-
Picking colonies of T7-rbs-merr and shaking at 37℃ overnight.
+
Do the 4th point mutation PCR step.
-
 
+
===7.23===
-
===7.24===
+
Identify the plasmid using agarose gel electrophoresis.
-
Problems existed in our experiment: The PCR reaction product cannot be verified. New primers forward (3C and 3Plus) for mbp fragments are designed.
+
Send the plasmid DNA for sequencing.
-
 
+
Put the rbs+agn43 into plasmid by ligation.
-
1. PCR reaction for mbp fragments using dissolved primers.
+
===7.29===
-
 
+
Attend the group seminar.
-
For                    1uL
+
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.
-
Rev                    1uL
+
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.
-
Easymix                10uL
+
Identify the T7+rbs+merT+rbs+merP+rbs+merC by electrophoresis.
-
 
+
-
template                0.2uL
+
-
 
+
-
ddH2O                up to 20 uL
+
-
 
+
-
2. Miniprep of T7-rbs-merr
+
-
 
+
-
3. Digestion and identification by Electrophoresis
+
-
 
+
-
EcoRI Buffer    1uL 
+
-
 
+
-
EcorRI        0.75uL
+
-
 
+
-
PstI          0.75uL
+
-
 
+
-
T7-rbs-merr  7.5uL
+
-
 
+
-
The band was not correct.
+
-
 
+
-
===7.25===
+
-
1. Electrophoresis PCR reaction product last day in 1% agarose gel.
+
-
 
+
-
2. Excise the gel slice and extract the mbp fragments.
+
-
 
+
-
3. DNA double digestion of mbp fragments for 3 hours: Xbal/XhoI for commercialization and XbaI/PstI for standardization.  
+
-
 
+
-
20uL reaction system:   
+
-
 
+
-
Buffer4    2uL 
+
-
 
+
-
Xbal      1.5uL
+
-
 
+
-
XhoI      1.5uL
+
-
 
+
-
Product  15uL
+
-
 
+
-
 
+
-
Buffer3    2uL 
+
-
 
+
-
XbaI      1.5uL
+
-
 
+
-
PstI      1.5uL
+
-
 
+
-
Product  15uL
+
-
 
+
-
2. DNA Purification.
+
-
 
+
-
3. ligation for 1 hour
+
-
 
+
-
mbp(XbaI/XhoI)    6uL
+
-
 
+
-
pET-21a            2uL
+
-
 
+
-
10x ligation buffer    1uL
+
-
 
+
-
T4 DNA Ligase      1uL
+
-
 
+
-
 
+
-
mbp (Xbal/PstI)          6uL
+
-
 
+
-
B0034 (rbs)              2uL
+
-
 
+
-
10x ligation buffer        1uL
+
-
 
+
-
T4 DNA Ligase            1uL
+
-
 
+
-
===7.26===
+
-
Transformation of ligation mixure.
+
-
 
+
-
Name of plates were: 3C-com, 3Plus-com, 3C-SD, 3plus-SD
+
-
 
+
-
*com means commercialization and SD means standardization
+
-
 
+
-
===7.27-7.28===
+
-
No colonies formation of 3C-com and 3plus-com
+
-
 
+
-
1. Picking 18 colonies from 3C-SD and 3plus-SD(mbp_SD) and shaking at 37℃ for 10 hours.
+
-
 
+
-
2. Miniprep mbp_SD
+
-
 
+
-
3. Digestion and identification by Electrophoresis
+
-
 
+
-
EcoRI Buffer    1uL 
+
-
 
+
-
EcorRI        0.75uL
+
-
 
+
-
PstI          0.75uL
+
-
 
+
-
mbp_SD      7.5uL
+
-
 
+
-
Sequence.  
+
-
 
+
-
(The result is confirmed to be incorrect.)
+
-
 
+
===7.30===
===7.30===
-
Template of pASK-mbp is verified to have mutagenesis; therefore we decide to construct mbp by linking 2 merr fragments together.
+
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.  
-
1. PCR reaction for six merr fragments: N_B and B_X; SD(Xbal)_B and B_SD(PstI); SD(EcoRI)_B and B_SD(PstI).
+
Do the transformation.
-
 
+
-
10x buffer              5uL
+
-
 
+
-
ddNTP Mixture          5uL
+
-
 
+
-
For                    1uL
+
-
 
+
-
Rev                    1uL
+
-
 
+
-
Template plasmid        0.5uL
+
-
 
+
-
Polymerase              1uL
+
-
 
+
-
ddH2O                up to 50 uL
+
-
 
+
-
2. Electrophoresis PCR reaction system in 1% agarose gel to identification.
+
-
 
+
-
3. DNA purification from reaction mixture.
+
-
 
+
-
4. DNA double digestion for 3 hours.
+
-
 
+
-
20uL reaction system:   
+
-
 
+
-
N_B:
+
-
 
+
-
Buffer3    2uL 
+
-
 
+
-
NdeI      1.5uL
+
-
 
+
-
BamHI    1.5uL
+
-
 
+
-
Product  15uL
+
-
 
+
-
B_X:
+
-
 
+
-
Buffer3    2uL 
+
-
 
+
-
BamHI    1.5uL
+
-
 
+
-
XhoI      1.5uL
+
-
 
+
-
Product  15uL
+
-
 
+
-
SD(Xbal)_B:
+
-
 
+
-
Buffer3    2uL 
+
-
 
+
-
XbaI      1.5uL
+
-
 
+
-
BamHI    1.5uL
+
-
 
+
-
Product  15uL
+
-
 
+
-
B_SD(PstI):
+
-
 
+
-
Buffer3    2uL 
+
-
 
+
-
BamHI    1.5uL
+
-
 
+
-
PstI      1.5uL
+
-
 
+
-
Product  15uL
+
-
 
+
-
SD(EcoRI)_B:
+
-
 
+
-
EcoRI Buffer    2uL 
+
-
 
+
-
EcorRI      1.5uL
+
-
 
+
-
BamHI      1.5uL
+
-
 
+
-
Product      15uL
+
-
 
+
-
5. DNA purification after the double digestion.
+
-
 
+
-
6. ligation of 2 merr fragments into the vector for 6 hours.
+
-
 
+
-
Insert1              3uL
+
-
 
+
-
Insert2              3uL
+
-
 
+
-
Vector              2uL
+
-
 
+
-
10x ligation buffer      1uL
+
-
 
+
-
T4 DNA Ligase        1uL
+
-
 
+
-
mbp-Com: Inserts: N_B and B_X; vector: pET-21a(NdeI/Xhol)
+
-
 
+
-
mbp-SD: Inserts: SD(Xbal)_B and B_SD(PstI), vector:B0034(SpeI/PstI)
+
-
 
+
-
mbp: SD(EcoRI)_B and B_SD(PstI), vector: pSB1K3 (EcoRI/PstI)
+
-
 
+
-
7. Transformation
+
-
 
+
===7.31===
===7.31===
-
No colonies formation of mbp-Com.
+
Digest 2-2E constitutive promoter with EcoRl and Spel. Digest rbs+agn43 with EcoRl and Xbal.
-
 
+
Connect 2-2E promoter with rbs+agn43.
-
1. Picking colonies from mbp-SD and mbp, shaking at 37℃ for 10 hours.
+
-
 
+
-
2. Miniprep of mbp-SD and mbp
+
-
 
+
-
3. Miniprep of B0034
+
-
 
+
-
4. Double digestion for 3 hours.
+
-
 
+
-
Buffer2    2uL 
+
-
 
+
-
SpeI    1.5uL
+
-
 
+
-
PstI      1.5uL
+
-
 
+
-
B0034(rbs)    5uL
+
-
 
+
-
ddH2O  up to 20 uL
+

Revision as of 10:53, 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

Mon Tue Wed Thu Fri Sat Sun
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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
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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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