The goal is this step is to ligate double stranded linkers to each end of the DNA fragments you created with restriction enzymes. The linkers provide PCR primer binding sites for subsequent steps.. It is a pretty cool idea!
1st step is to make the double stranded linkers.. This is easy. Simple mix equimolar amounts of each primer, heat to 95 degrees, and cool slowly. They are designed such that they self-anneal.
Next is the ligation, which is also very easy!
| NEB 10X Ligase Buffer | 1uL |
| NEB T4Ligase | 2uL |
| ds SuperSNX Linkers | 7uL |
To this mix, add 21uL of your restriction enzyme digest, and incubate at 16 degrees overnight..
There is a PCR check on this step- using one of the SNX primers. What you should see is a continuous smear from >1000bp to 300bp. If there is a smear, then you know that the double stranded primers have been successfully been ligated to the fragments generated by the restriction enzyme digest…
Here is my image:
The image quality is poor- clicking on the image improves it.. something about the way blogger compresses images I suppose…
Anyway, the image indicates that up till now, the process is going smoothly.
1 response so far ↓
1 Rich Lawler // Sep 28, 2007 at 8:16 pm
What protocol are you using? If you can find the protocol by Hammond et al., this is a good one that walks you through the process. It is good to look at even if you aren’t using it.
Here’s the reference:
Hammond RL, Saccheri IJ, Ciofi C, Coote T, Funk SM, McMillan WO, Bayes MK, Taylor E, Bruford MW. 1998. Isolation of microsatellite markers in animals. In: Molecular
tools for screening biodiversity.
London: Chapman and Hall. p 279–285.
Also, when you get to the enrichment phase (where you use oligo probes of CA-CA-CA in order to find msats,) order them with a 3′ chain terminator. This prevents you from getting concatamers during the PCR enrichment phase (if this makes sense).
Here’s the reference:
Koblizkova A, Dolezel J, Macas J. 1998. Subtraction of 3′ modified oligonucleotides eliminates amplification artifacts in DNA
libraries enriched for microsatellites. Biotechniques 25:32–38.
I don’t envy you, but good luck!
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