User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/04/06: Difference between revisions
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| colspan="2" | *For low yield DNA, use up to 25 μL; decrease dH<sub>2</sub>O accordingly.<br>Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at 37°C for 30 minutes. | | colspan="2" | *For low yield DNA, use up to 25 μL; decrease dH<sub>2</sub>O accordingly.<br>Mix the reaction(s) thoroughly by flicking the tube.<br>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''' | '''Separate the fragments via gel electrophoresis and purify the fragments''' | ||
[[Image: | [[Image:BD111-T7-Gel-Behzad 6-4-12.jpg|thumb|350px|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. | # 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. | # 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. | ||
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# 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). | # 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. | # 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. | ||
{| class="wikitable" width=400px align="left" | |||
| || Conc.(ng/μl) ( || (A260/A280) | |||
|- | |||
| RBS-PcTF insert || 7.606 || 2.235 | |||
|- | |||
| Vector BBa_I712074 (T7-Strong)|| 15.606 || 1.814 μL | |||
|- | |||
| Vector BBa_I719005 (T7) || 16.38 || 2.104 μL | |||
|- | |||
|} |
Revision as of 14:00, 7 April 2012
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06/04/12Assemblies
> Digests (Fermentas FD)
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
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