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iGEM Training Course

iGEM Training Course. 2009. ~ Experiments and lab rules ~. Wei-Chao Liao. Biobrick construction. (Wet-lab). Bacteria culture and plating Purification of genomic and plasmid DNA Primer design PCR Purification of PCR products DNA DNA digestion and ligation Transformation of target vector

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iGEM Training Course

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  1. iGEM Training Course 2009 ~ Experiments and lab rules ~ Wei-Chao Liao

  2. Biobrick construction (Wet-lab) • Bacteria culture and plating • Purification of genomic and plasmid DNA • Primer design • PCR • Purification of PCR products DNA • DNA digestion and ligation • Transformation of target vector • Confirm the results

  3. Preparation of medium and plate • LB or M9 (depend on bacteria and exp. condition) Batch makes about 20 plates: 1. Add 150 mL of ddH2O to a graduated cyclindar 2. Weigh out 5.0 g tryptone, 2.5 g yeast extract, 5.0 g NaCl and 7.5 g agar 3. Mix powder well to bring into solution 4. Add ddH2O to total volume of 300 mL and transfer to 500 mL flask 5. Label with autoclave tape and autoclave for 20 min 6. Let agar cool to ~55 ℃ 7. Antibiotics (agar: temperature and concentration) 8. Storage at 4 ℃

  4. DNA purification kits • Plasmid DNA kit • Genomic DNA kit • Gel extraction kit • PCR cleanup kit

  5. Primer design tool • Vector NTI v9.0

  6. Primer design notes • Primer for gene (CDS) cloning - May not be a perfect design, - RE site, - No. of addition base, • Primer sequence check by NCBI blastn • Test PCR annealing condition (Tm.) • To avoid any hairpin, palindrome and repeat

  7. Primer design notes Purification by PAGE is recommended, if length more than 40 bp. http://bioweb.uwlax.edu/GenWeb/Molecular/seq_anal/primer_design/primer_design.htm

  8. Polymerase chain reaction (PCR) Template (conc.) Primer pair (10ρm & Tm.) DNA polymerase (pfu) dNTP (2.5 mM) + dNTP

  9. Cloning notes • Vector : insert • Vector concentration • Insert length • Total plasmid length • Vector: • Selection marker • Strongly promoter • Replication origin • MCS http://bio.classes.ucsc.edu/bio20L/info/content/molbio2/pks.gif

  10. Lab rules • 垃圾分類： • 生物感染性垃圾 • 一般垃圾 (丟外面) • 有機廢液回收 PS： 電腦桌 VS. 實驗桌： 文具用品…

  11. Lab rules • 冷凍低溫冰箱 • 恆溫培養箱 • Pipette • Bench • Tips

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