User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/04/06

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  1. BD112 (final assembly 1): BD111/(X/P)/1138 + BBa_I712074/(S/P)46
  2. BD113 (final assembly 2): BD111/(X/P)/1138 + BBa_I719005/(S/P)23
BD112 and BD113 assembly

> Digests (Fermentas FD)

Plasmid DNA 20.0 μl*
Fermentas FastDigest enzyme 1 1.0 μl
Fermentas FastDigest enzyme 2 1.0 μl
10x FastDigest buffer + green loading dye 3.0 μl
dH2O 5.0 μl
  30.0 μl total
*For low yield DNA, use up to 25 μL; decrease dH2O accordingly.
Mix the reaction(s) thoroughly by flicking the tube.
Incubate at 37°C for 30 minutes.
  • Two tubes of BD111 cut with (X/P), One tube of BBa_I712074 (T7 strong promoter) and one tube of BBa_I719005 (T7 promoter) cut with (S/P).

Separate the fragments via gel electrophoresis and purify the fragments

White dashed lines border where the gel was cut to excise (1 & 2)insert fragment of PcTF-RBS cut with (X/P)), (3) vector fragment (BBa_I712074, T7 strong promoter with plasmid backbone: pSB1AK8)and (4)vector fragment (BBa_I719005, T7 promoter with plasmid backbone: pSB1A2)- vector fragments (3 & 4)cut with (S/P).
  1. Make a 0.8% gel: add 0.5 g agarose to ~60 ml 1x TAE buffer in a glass flask.
  2. Mix by swirling and microwave for 40 seconds. Mix by swirling again (to eliminate air pockets and prevent boiling-over) and microwave for 40 seconds.
  3. Set up a gel mold and comb. Make sure the teeth are the right size to hold 30 μL of sample.
  4. Add 6 μl of 10 mg/ml ethidium bromide (etBr) to the agarose for a final concentration of ~0.8 μg/mL etBr. Mix by swirling (avoid making bubbles).
  5. Pour the gel into the gel mold. Allow it to cool until it becomes opaque.
  6. Fill a gel electrophoresis chamber with 1x TAE.
  7. Remove the comb from the gel and carefully submerge the gel into the filled electrophoresis chamber.
  8. Carefully pipette 15 μL pre-made 1 kb ladder mix into the first empty well and the DNA samples into the other empty wells.
  9. Connect the electrical leads so that the positive end is at the bottom (DNA migrates to the positive end). Run the gel at 100 V.
  10. Stop the gel when the yellow dye (Orange G) reaches the desired place on the gel (~1 hr.).
  11. Remove the gel from the chamber and photograph under UV light.
  12. Use a scalpel to cut the appropriate sized band(s) from the gel, place each gel slice in a 1.5 mL tube, and purify the DNA with Zymoclean Gel DNA Recovery Kit(Follow the protocol inside the kit box).
  13. Measure the concentration of the purified fragment samples with a Biotek Take3 plate from the Stabenfeldt lab. Record the absorbance (A260), purity (A260/A280), and concentration (ng/μl) for each sample.
  • Insert RBS-PcTF + (S/X) : 15 + 1123 + 6 = 1144 bp
  • Vector T7 strong promoter (BBa_I712074) 46 bp plasmid backbone is pSB1AK8 size: 3426 bp → 3426 + 46 = 3472 bp
  • Vector T7 promoter (BBa_I719005) 23 bp plasmid backbone is pSB1A2 size: 2079 bp → 2079 + 23 = 2102 bp
  Conc.(ng/μl) ( (A260/A280)
RBS-PcTF insert 7.606 2.235
Vector BBa_I712074 (T7-Strong) 15.606 1.814
Vector BBa_I719005 (T7) 16.38 2.104