User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/04/06: Difference between revisions
From OpenWetWare
No edit summary |
No edit summary |
||
(3 intermediate revisions by the same user not shown) | |||
Line 31: | Line 31: | ||
| 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). | *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''' | ||
Line 48: | Line 48: | ||
# 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. | ||
* 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 | |||
{| class="wikitable" width=400px align="left" | {| class="wikitable" width=400px align="left" | ||
Line 54: | Line 58: | ||
| RBS-PcTF insert || 7.606 || 2.235 | | RBS-PcTF insert || 7.606 || 2.235 | ||
|- | |- | ||
| Vector BBa_I712074 (T7-Strong)|| 15.606 || 1.814 | | Vector BBa_I712074 (T7-Strong)|| 15.606 || 1.814 | ||
|- | |- | ||
| Vector BBa_I719005 (T7) || 16.38 || 2.104 | | Vector BBa_I719005 (T7) || 16.38 || 2.104 | ||
|- | |- | ||
|} | |} |
Revision as of 14:16, 7 April 2012
PcTF Subcloning in E. coli | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||
06/04/12Assemblies
> Digests (Fermentas FD)
Separate the fragments via gel electrophoresis and purify the fragments
|