Hi!

I have been growing colonies from mixed gut samples, and while most amplified easily, I am still left with quite a few that managed to avoid amplifying. I tried regular Taq with bacterial 16s (27f (a mixture of YM, BIF, BOR, CHL)/907r), Archeal 16s (340f/1000r), and 18s for yeasts. Tried with 1ul and 5 ul of template. Tried repicking the colonies, bead beating them (.1mm +.5mm glass beads). I can't seem to get ANY bands. I will be trying gram staining today, but since most of these were anaerobic samples, I am thinking that they might now be degraded since they have been out in the aerobic fridge for a few days now.

So, what is you best personal trick to get those complex samples to amplify?? DMSO? BSA? More MgCl2? Lower annealing temp (which I can't do with archaea, I'm already at the lowest recommended for these primers to stay specific enough for archaea). HiFi Taq?

My thermocycler programs are as follow:

Bacterial 16s:

1. 95° for 3 min

2. 95° for 30 sec

3. 48° for 30 sec

4. 72° for 1 min

5. Cycle to step 2 for 34 more times

6. 72° for 10 min

Yeasts 18s:

1. 95° for 3 min

2. 95° for 30 sec

3. 50° for 30 sec

4. 72° for 45 sec

5. Cycle to step 2 for 34 more times

6. 72° for 5 min

Archaeal 16s:

1. 98° for 3 min

2. 95° for 30 sec

3. 57° for 30 sec

4. 72° for 1:00 min

5. Cycle to step 2 for 34 more times

6. 72° for 5 min

My basic recipe for a 25ul reaction:

2.5ul 10x regular buffer (that comes with my Taq)

0.5ul dNTPs (2.5mM if I recall correctly)

1.5ul MgCl2

1ul forward buffer (10mM)

1ul reverse buffer (10mM)

1 or 5 ul DNA template

18.4 or 14.4 ddH2O

0.1ul Taq (5u/ul)

Any help will be greatly appreciated!

Thanks :)