blunt-end ligation - (Nov/16/2008 )
Can anyone help me solve my problem about blunt-end ligation? I have a vector which is about 11Kb and an insert that is 1Kb. I'm expecting 4 bands with sizes of 8Kb, 2.5Kb, 300bp and 49 bp respectively. I find it hard to deal with them because everytime I checked the plasmid with RE digestion, two lower bands were always missing 
. But, I usually obtained the 8Kb and 2.5Kb correctly. I fill the gaps and used SAP prior to ligation. Can you please suggest what to do better to improve this condition? Thank you.
hannah_jlb
-hannah_jlb-
QUOTE (hannah_jlb @ Nov 17 2008, 05:51 AM)
Can anyone help me solve my problem about blunt-end ligation? I have a vector which is about 11Kb and an insert that is 1Kb. I'm expecting 4 bands with sizes of 8Kb, 2.5Kb, 300bp and 49 bp respectively. I find it hard to deal with them because everytime I checked the plasmid with RE digestion, two lower bands were always missing . But, I usually obtained the 8Kb and 2.5Kb correctly. I fill the gaps and used SAP prior to ligation. Can you please suggest what to do better to improve this condition? Thank you.
hannah_jlb
. But, I usually obtained the 8Kb and 2.5Kb correctly. I fill the gaps and used SAP prior to ligation. Can you please suggest what to do better to improve this condition? Thank you.hannah_jlb
Hannah,
Can you post my reply to your PM about this topic here?
Clare
-Clare-
QUOTE (hannah_jlb @ Nov 17 2008, 04:51 PM)
Can anyone help me solve my problem about blunt-end ligation? I have a vector which is about 11Kb and an insert that is 1Kb. I'm expecting 4 bands with sizes of 8Kb, 2.5Kb, 300bp and 49 bp respectively. I find it hard to deal with them because everytime I checked the plasmid with RE digestion, two lower bands were always missing . But, I usually obtained the 8Kb and 2.5Kb correctly. I fill the gaps and used SAP prior to ligation. Can you please suggest what to do better to improve this condition? Thank you.
hannah_jlb
. But, I usually obtained the 8Kb and 2.5Kb correctly. I fill the gaps and used SAP prior to ligation. Can you please suggest what to do better to improve this condition? Thank you.hannah_jlb
Unless you are cutting large amounts of vector, and running the digest on a high percentage gel, you probably won't see the 300 and 49 bp fragments (it will also not interact well with EtBr). 1ug of an 11 kb vector will only have 27 ng of the 300 bp fragment and 4 ng of the 49 bp.
-swanny-