November 1, 2011 at 3:13 pm #1479
I have been working on amplifying bacteria from the guts of c.elegans.
My protocol is as follows:
– extract gut from nematode (yes the gut is less than a 1 mm wide and about 3-5 mm long)
– freeze for 10 mins (at -80) to lyse the cells
– run a 5% prot k treatment
– add reagents for PCR as follows:
2.5-2.9 uL of MgCl2
3 uL of Hot Start Buffer
3 uL of each primer (forward and reverse, im using 1492R and 27F)
1 uL of dntps at 6 uM conecntration
14.44-14.04 uL of H2O (altered based on mgcl2 amount)
2.5 uL (approx) of DNA sample
30 uL total volume
using an annealing temp of 54 C and running a standard protocol with 35 cycles. also added an extra 5min at 95 C, at the start before the loop. [even added extra 30 seconds (1.5 min total) to extension cycle but still didnt work]
My problem is that I get NO amplification. The positive control shows up (using extracted genomic DNA from streak) but I see no bands for my samples. Occasionally I see one or two (out of 5) but it is extremely inconsistent.
I have tried running a MgCl2 gradient (2.5, 2.7, 2.9, 3.1 etc…) to no avail.
I’ve heard that my sample could be amplifying but that its still two low to see a band. I’ve quantified the product via Qubit and they are substantially (10 fold) more concentrated than my -control (all reagents but no template). For some reason however doing a nested PCR doesn’t seem to produce results.
I have tried to proceed with the spotty results that I get (TOPO cloning vector) but unfortunately there seemed to be no insert in the vectors after amplifying 20-30 different colonies and attempting the whole cloning a second time.
The price of the cloning is far to expensive for me to keep attempting and I was hoping that at some point I would get my guts to amplify at least 80% of the time.
Anybody have any suggestions as to what I might to do improve the amplification and/or verify that I actually have product even if the bands are not showing up.
I have been working on this for almost a year and it is very discouraging. Please help 🙁
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