Northern blotting - How to chose a probe for Norther blot? (May/20/2008 )
I have to perform my first time Northern blotting. Although I looked at the past postings about this issue, I couldn't quite tell how to choose the probe for Northern blotting. Could any one help me?
The RNA of my interest is Grp78.
Should I use order made probes by looking at the sequence of Grp78?
-mgs1021-
QUOTE (mgs1021 @ May 20 2008, 02:53 PM)
I have to perform my first time Northern blotting. Although I looked at the past postings about this issue, I couldn't quite tell how to choose the probe for Northern blotting. Could any one help me?
The RNA of my interest is Grp78.
Should I use order made probes by looking at the sequence of Grp78?
The RNA of my interest is Grp78.
Should I use order made probes by looking at the sequence of Grp78?
1. You want to make about 300-500 bp probe.
2. Avoid repeat sequences
3. If Grp78 sequence has similarity with other members of the family, and you want to detect only Grp78, try to select the most unique stretch of sequence. Avoid areas that are similar.
4. The area that you choose should be easily PCRable. Meaning, if you are planning to do RT-PCR for generating the probe, you need to select two good primers. If you are planning to cut a plasmid already containing your cDNA, try to choose area between two unique restriction sites.
5. Generally speaking, try to have GC% about 50 or more. But that depends.
-cellcounter-
thank you for replying. i will reconsider it again.
QUOTE (cellcounter @ May 20 2008, 05:00 PM)
QUOTE (mgs1021 @ May 20 2008, 02:53 PM)
I have to perform my first time Northern blotting. Although I looked at the past postings about this issue, I couldn't quite tell how to choose the probe for Northern blotting. Could any one help me?
The RNA of my interest is Grp78.
Should I use order made probes by looking at the sequence of Grp78?
The RNA of my interest is Grp78.
Should I use order made probes by looking at the sequence of Grp78?
1. You want to make about 300-500 bp probe.
2. Avoid repeat sequences
3. If Grp78 sequence has similarity with other members of the family, and you want to detect only Grp78, try to select the most unique stretch of sequence. Avoid areas that are similar.
4. The area that you choose should be easily PCRable. Meaning, if you are planning to do RT-PCR for generating the probe, you need to select two good primers. If you are planning to cut a plasmid already containing your cDNA, try to choose area between two unique restriction sites.
5. Generally speaking, try to have GC% about 50 or more. But that depends.
-mgs1021-