Team:Tsinghua/experiments

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Experiment Records

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Our Experiments were carried out by the following nine groups which are somewhat independent from each other. Here come the Groups and Their Tasks.

Group 1A:Landing pad (LP) construction and Insertion with Tranditional Att Recomination method(Promoter Inside)

Group 1B:Landing pad (LP) construction and Insertion with Innovational Att Recomination method(Promoter Outside)

Group 1C:Landing pad (LP) construction and Insertion: Inducement and Recombination

Group 2A:Donor Plasmid Construction: Two Fragments Construction (Three Fragments Ligation)

Group 2B:Donor Plasmid Construction: Traditional Four Fragments Construction

Group 2C:Donor Plasmid Construction: Four Fragments Construction with Dra III

Group 2D:Donor Plasmid Construction: Four Fragments Construction with Novel Methods

Group 3:Helper Plasmid (HP) Insertion, Donor Plasmid Insertion, Recombination Induction & Removal of HP

Group 4:Screening Strategy









Protocols

Here are our Experimental Protocols in PDF version. You're advised to view in Acrobat or Adobe Reader which you can download free from the Home Website of Adobe® Company, for other viewers we can not guarantee the right format of viewing. Those files are encrypted to keep copyrights, if you want to print them out of copy to your documents, to share with others our ideas and methods, you are welcome to ask us for the unencrypted files definitely for free~

Molecular Cloning
THUProtocol_1-1_Isolatio_of_plasmid_DNA

THUProtocol_1-2_DNA_Gel_Extraction

THUProtocol_1-3_Restriction_Enzyme_Digestion

THUProtocol_1-4_Ligation_of_DNA_Fragments

THUProtocol_1-5_PCR

THUProtocol_1-6_Preparation_of_Competent_Cell_for_Electro_Transformation

THUProtocol_1-7_Chemical_Transformation_of_Recombinant_DNA

THUProtocol_1-8_Electro_Transformation_of_Recombinant_DNA

Protein Isolation and Identification
THUProtocol_2-1_Protein_Isolation_for_Prokaryotes

THUProtocol_2-2_Protein_Identification_SDS-PAGE

THUProtocol_1-5_PCR

Results

Group 2(b)
1. PCR: Amplication eGFP, mCherry, Kanamycin resistant gene and Chlr four genes separatly from PIB-eGFP, PBS34, PKD13 and PKD3 plasmids.

Fan1.JPG Fan2.JPG


2. pUC19 double digestion for eGFP and Chlr, ligate with digested eGFP and Chlr separately. PCR to detect pUC19+eGFP and pUC19+ChlrPCR. All 12 samples are positive.

THU-Fan3.JPG


3. Double digest PE1 and mCherry with SalI and BamHI, double digest PC1 and Kan with KpnI and BamHI, ligate and PCR to detect. No positive results exist among PC1+K.

THU-Fan4.JPG


4. Double digest PEM2 and Kan with KpnI and BamHI as well as PC1. Ligate and using PCR to detect with universal primers. PEM2+K 1 and 5 are positive.

THU-Fan5.JPG


5. Double digest PEMK1 and Chlr with KpnI and EcoRI. Ligate and using PCR to detect with universal primers. PEMK1+Chlr 3-5 are positive.

THU-Fan6.JPG

Group 2(c)
1. 2010.7.31 PCR result for eGFP, Kan and Chlr. No specific target bands appeared in the photograph. Besides, dimerization of primers is severe.

THU-hdy-1.JPG


2. 2010.8.16 RCR result for eGFP, mCherry, Kan and Chlr.

THU-hdy-2.JPG


3. Result : plasmid digestion with EcoR I and Pst I.

THU-hdy-3.JPG


4. Identification: After digestion, ligation and transformation, plasmid is extracted and digested with EcoR I. The size of positive clone is about 2.7Kb while the negative is less than 2Kb. As the photograph shows, there was no positive clone.

THU-hdy-4.JPG


5. Identification: After digestion, ligation and transformation, plasmid is extracted and digested with EcoR I. The size of positive clone is about 2.7Kb while the negative is less than 2Kb. As the photograph shows, two clones, whose size is about 3Kb (plasmid+ Chlr), are positive.

THU-hdy-5.JPG


6. As no positive clone for plasmid-eGFP-mCherry-Kan is picked, we have strong doubt about the efficiency of ligation of multiple fragments. Thus, PCR is conducted to confirm whether the ligation reaction took place in the system. Upstream primer of eGFP and downstream primer of Kan are used in PCR. Bands with the size of about 2.5Kb is hardly seen in the gel photograph.

THU-hdy-6.JPG