Hi everyone,
I will keep my question brief and specific to (hopefully) generate interest, and if you want me to give more background I'm happy to do so.
I am using a modified AFLP protocol to reduce my sample (amphibian) genome size. I hope to sequence 2-5 individuals with this RRL on the 454 Junior platform. My problem is the last step, the selective amplification. For those of you familiar with AFLPs, the protocol is -
1. One-step digestion/ligation - I tried 2 different enzyme combos: EcoRI/MseI, HinDIII/MspI. Adapters are ~13bp.
2. Pre-Amplification - Using EcoRI and MseI primers, ~17bp.
3. Gel size selection and purification - I'm using 1xTBE buffer, 1% gel, 70V (on large gel well), regular agarose (says best for fragments >1kb, but we routinely use it for smaller fragments). I'm purifying with Qiagen gel extraction kit.
4. Selective Amplification - Using longer primers with EcoRI or MseI sequence + MID barcode + fusion primer sequence.
See the 3 gels attached. When I select moderate size fragments, the resulting amplicons are always tiny (1-200bp). Selected size has no relationship with amplicon size. In other words, makes no difference whether I select 500-600 or 900-1000, always comes out 200. We need 400bp. I found one paper describing a downward size bias for my particular taq - Phusion HF - however it's my understanding this taq is standard for 454 library prep. Also, you'll notice wavy bands on all gels - I'm using Gel Red for staining.
What's going on?! Thanks in advance.
I will keep my question brief and specific to (hopefully) generate interest, and if you want me to give more background I'm happy to do so.
I am using a modified AFLP protocol to reduce my sample (amphibian) genome size. I hope to sequence 2-5 individuals with this RRL on the 454 Junior platform. My problem is the last step, the selective amplification. For those of you familiar with AFLPs, the protocol is -
1. One-step digestion/ligation - I tried 2 different enzyme combos: EcoRI/MseI, HinDIII/MspI. Adapters are ~13bp.
2. Pre-Amplification - Using EcoRI and MseI primers, ~17bp.
3. Gel size selection and purification - I'm using 1xTBE buffer, 1% gel, 70V (on large gel well), regular agarose (says best for fragments >1kb, but we routinely use it for smaller fragments). I'm purifying with Qiagen gel extraction kit.
4. Selective Amplification - Using longer primers with EcoRI or MseI sequence + MID barcode + fusion primer sequence.
See the 3 gels attached. When I select moderate size fragments, the resulting amplicons are always tiny (1-200bp). Selected size has no relationship with amplicon size. In other words, makes no difference whether I select 500-600 or 900-1000, always comes out 200. We need 400bp. I found one paper describing a downward size bias for my particular taq - Phusion HF - however it's my understanding this taq is standard for 454 library prep. Also, you'll notice wavy bands on all gels - I'm using Gel Red for staining.
What's going on?! Thanks in advance.
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