Cloning to make rna probes for in situ

[ah535]

Our lab would like to insert our pcr products into a plasmid, insert the plasmid into a bacteria, store it, then preform a reverse transcription on the inserted gene so that we can use the rna produced from the product as a probe for in situ.

1. Is this a good way to create your own probe?
2. If so what supplies will be need? We have everything to do pcr and gel extractions and that is it.
3. What literature/protocols are there on this topic so that I can become familiar with the best way to do this.

Thank you so much for your help.

[mmorgan]

It sounds like you just want to generate anti-sense RNA probes for hybridization. This is a very standard technique, you just need to clone your insert into a plasmid with phage promoter sequences (i.e. T7, T3). You can then purify and linearize your plasmid and do an in vitro transcription reaction using T7 or T3 RNA polymerase (you don't use reverse transcriptase as stated in your post).

Most molecular biology texts (i.e. Molecular Cloning: A lab manual) will give detailed protocols.

Another way to generate probes is to PCR amplify fragments with a T7/T3 site in one of the primers. In this method the PCR product can be used directly in the transcription reaction.

[ah535]

Thank you this is extremely helpful information.

[ah535]

One last question that I have is regarding my gel extracted product. I have a lot of salt carry over from using a mini-elute kit. Can I do an ethanol precipitation on a product that has already been gel extracted or is there a better way to desalt the product before ligation?

[mmorgan]

Originally Posted by ah535

One last question that I have is regarding my gel extracted product. I have a lot of salt carry over from using a mini-elute kit. Can I do an ethanol precipitation on a product that has already been gel extracted or is there a better way to desalt the product before ligation?

EtOH precipitation is a pretty good way to de-salt although this shouldn't really be necessary if the kit is decent. I always use the QIAEX II kit from Qiagen. This is the one with the glass bead slurry that you spin down. It seems to work much better than the column preps. Probably there is not that much DNA after a gel extraction so be careful with the pellet, or maybe add a little glycogen, I don't think this would interfere too much with the ligation.

[ah535]

I am sorry, I really don't know a lot about this topic. Could you expound on the second method. I thought the T7 was an RNA polymerase, but you said that we need a T7 site in our pcr product. I am guessing that we need to add some kind of promoter sequence to our already amplified product for the T7 RNA to bind to. Is there a common kit for this? Also, is there a way to check that your transcribed product is good? What are the pros/cons of the methods that you described.

Thank you for being patient.

[ah535]

After doing some reading in the Molecular Cloning manual I was able to answer all of my questions.