Berkeley LBL/JoyceCyano
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'''Contruction of pET3a Derivatives Containing ''chlD'' and ''chlI''''' | '''Contruction of pET3a Derivatives Containing ''chlD'' and ''chlI''''' | ||
| - | 1. Amplify | + | 1. Amplify ''Synecholystis Sp.'' gene and introduce restriction sites by [[Berkeley_LBL/PCRphusion|PCR (Using Phusion Polymerase)]], which allow cloning PCR fragments into pET3a |
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Latest revision as of 02:55, 27 October 2007
Contruction of pET3a Derivatives Containing chlD and chlI
1. Amplify Synecholystis Sp. gene and introduce restriction sites by PCR (Using Phusion Polymerase), which allow cloning PCR fragments into pET3a
| chlD | chlI | |
|---|---|---|
| length of gene fragment | 2031 b.p. | 1074 b.p. |
| Restriction sites introduced when amplified by PCR | NdeI, BglI | NdeI, BglI |
Synecholystis Sp. Genomic DNA 1 ul HF Buffer 10 ul dNTP 1 ul Primer Mix 5 ul Water 32.5 ul Phusion Polymerase 0.5 ul
| bchD | ||||||
| 1.Initial Denaturation | 2.Denaturation | 3.Annealing | 4.Extension | 5.Repeat step 2 to 4 for 30 times | 6.Final Extension | |
| 98 °C | 98 °C | 62 °C | 72 °C | 72 °C | 4 °C | |
| 30 sec | 8 sec | 30 sec | 70 sec | 10 min | forever |
| bchI | ||||||
| 1.Initial Denaturation | 2.Denaturation | 3.Annealing | 4.Extension | 5.Repeat step 2 to 4 for 30 times | 6.Final Extension | |
| 98 °C | 98 °C | 64 °C | 72 °C | 72 °C | 4 °C | |
| 30 sec | 8 sec | 30 sec | 40 sec | 10 min | forever |
2. Do DNA Gel Electrophoresis to confirm that the length of the PCR fragments are the same as the corresponding target gene fragments.
3. Remove any leftover PCR enzyme in the samples by PCR Clean Up/Purification.
4. Digestion for PCR Products using specific restriction enzymes
| chlD | NdeI, BglI |
|---|---|
| chlI | NdeI, BglI |
4. Digested chlD and chlI are subjected to Gel Extraction to ensure that only pure, digested DNA is obtained and before doing ligation.
5. Ligate digested and purified PCR fragments with digested pET3a by Ligation yielding plasmids of pETBCHD and pETBCHI.
6. Use small portion of the plasmids to do Analytic Digestion.
7. Do KCM Competent Cell Transformation to subclone plasmids into DH10B competent cells, which have been done by KCM Competent Cell Production.
8. Pick colonies and innoculate in 2ml LB + 2ul Carbencilin in 37°C shaker overnight.
9. Extract plasmid DNA from bacteria by [[Berkeley_LBL/Miniprep|Miniprep].
10. Confirm that the plasmid DNA contains the right construct by Digestion for Miniprepped DNA and Analytic Digestion.
11. Do KCM Competent Cell Transformation to subclone plasmids into BL21, which have been done by KCM Competent Cell Production.
Protein Expression
Protein Analysis
