- BD112 (final assembly 1): BD111/(X/P)/1138 + BBa_I712074/(S/P)46
- BD113 (final assembly 2): BD111/(X/P)/1138 + BBa_I719005/(S/P)23
> 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).
- Make a 0.8% gel: add 0.5 g agarose to ~60 ml 1x TAE buffer in a glass flask.
- 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.
- Set up a gel mold and comb. Make sure the teeth are the right size to hold 30 μL of sample.
- 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).
- Pour the gel into the gel mold. Allow it to cool until it becomes opaque.
- Fill a gel electrophoresis chamber with 1x TAE.
- Remove the comb from the gel and carefully submerge the gel into the filled electrophoresis chamber.
- Carefully pipette 15 μL pre-made 1 kb ladder mix into the first empty well and the DNA samples into the other empty wells.
- 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.
- Stop the gel when the yellow dye (Orange G) reaches the desired place on the gel (~1 hr.).
- Remove the gel from the chamber and photograph under UV light.
- 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).
- 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