pBluescript cloning of PCR products - (Aug/29/2006 )

I'm using universal bacterial primers to amplify from environmental samples so I'm generating mixed PCR products. I want to be able to (cheaply) clone my PCR products so I can sequence and identify my bacterial species. I've engineered two different restriction sites into my primers.

1) Do I need two restriction sites since the direction of my insert doesn't matter?

I have pBluescript in XL1-Blue cells. I'll grow them overnight in LB with ampicillin and do minipreps. I haven't done much cloning in the past. Does anyone want to give a newbie (detailed) instructions on what to do next?

---

Just a bit of sharing, if the direction of your insert is not your concern, then you do not need the retriction sites.

I'm starting cloning as well, I grow them overnight in LB/ampilicilin/IPTG-XGal plates. Then I do a Blue-White screening, picking up single clone that I'm interested (Whites) and grow them again on to a new plate of agar (so that I have more to work with later). Which I later screen them again by PCR and sequence them.

Not sure that's what you need. Hope it helps.

---