742
edits
DNA ligation: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
no edit summary
Barry Canton (talk | contribs) |
No edit summary |
||
| Line 12: | Line 12: | ||
[[BE.109:DNA engineering/DNA ligation and bacterial transformation|BE.109:DNA ligation]] -- A ligation protocol for classroom use in a laboratory class taught at MIT. Uses T4 DNA ligase but has interesting tips and tricks. | [[BE.109:DNA engineering/DNA ligation and bacterial transformation|BE.109:DNA ligation]] -- A ligation protocol for classroom use in a laboratory class taught at MIT. Uses T4 DNA ligase but has interesting tips and tricks. | ||
==Factors Affecting Efficiency== | |||
A protocol analysis experiment for a typical DNA ligation (7.2 kb vector + 0.6 kb insert, sticky ends) gave optimal ligation efficiency when 50 ng of vector was ligated overnight at 16°C with a 2:1 insert:vector molar ratio and standard T4 ligase. Ligase was heat inactivated at 65°C for 20 mins before 2 µl (of 20 µl) was used to transform commercial heat-shock competent cells. | |||
Ligation efficiency was <b>marginally decreased</b> by | |||
#Doing a 1 hr ligation at room temperature | |||
#Using 100 ng vector | |||
#Using insert:vector molar ratios of 5:1 and 1:1 | |||
Ligation efficiency was <b>noticably decreased</b> (x100) by | |||
#Sticky end ligation with a larger insert (5.2 kb vector + 2.6 kb insert) | |||
#Blunt end ligation | |||
Ligation efficiency was <b>severely decreased</b> (x10000) by | |||
#Using DNA fragments that have been exposed to Ethidium Bromide and UV during the gel extraction procedure (<i>difficult to avoid but heartily recommended</i>) | |||
#Using the NEB Quick Ligation Kit | |||
==Discussion== | ==Discussion== | ||
