Journal:

Article Title: Preparing DNA Libraries for Multiplexed Paired-End Deep Sequencing for Illumina GA Sequencers

doi: 10.1002/9780471729259.mc01e04s20

Figure Lengend Snippet: Overview of DNA library preparation for deep sequencing. The purified genomic DNA is processed and modified as described in the basic protocols. The adapter-modified DNA fragments (library) are then enriched through PCR. Note the relative positions and characteristics (Inset) of the InPE1.0, InPE2.0 and the Index primers used during PCR. For multiplexed paired-end PCR, the same InPE1.0 and InPE2.0 primers are used, however, up to 12 different Index primers can be added to the enrichment reactions individually. Sequencing is performed in one direction through sequencing by synthesis, and then in the alternate direction as described in the background information. The complementary sequences are shaded darker.

Article Snippet: Materials list-behavior=simple prefix-word= mark-type=none max-label-size=0 Adapter-ligated DNA sample PCR Primer InPE1.0 (Illumina Cat. #PE400-1001) PCR Primer InPE2.0 (Illumina Cat. #PE400-1001) PCR Index primer (Illumina Cat. #PE400-1001) 5x HF Phusion buffer (NEB Cat. #F-540S) 10 mM dNTP (NEB Cat. #N0447S) Phusion DNA polymerase (NEB Cat. #F-540S) 6X gel loading dye with Gel Green (see recipe) 100 bp Marker (NEB Cat. #N3231S) 1X TBE (see recipe) 5% TBE DNA acrylamide gel (BioRad Cat. #161-1109) Sterile double-distilled water (nuclease-free) PCR thermal cycler to hold 200 μl PCR tubes with heated lid DNA acrylamide gel electrophoresis set-up QIAquick PCR Purification kit (Qiagen Cat. #28104) NanoDrop spectrophotometer (NanoDrop Model #ND100) list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Prepare the following reaction mix on ice in a new 200 μl PCR tube, making sure that the Phusion DNA polymerase is the last component to be added to the mix: list-behavior=enumerated prefix-word= mark-type=lower-alpha max-label-size=0 The PCR Index Primer used should be different for each strain DNA library constructed (e.g. strain 1 – Index Primer 1; strain 2 – Index Primer 2; strain 3 – Index Primer 3; etc) 175 ng Adapter-Ligated DNA sample 1 μl PCR Primer InPE1.0 for fragment amplification 1 μl PCR Primer InPE2.0 for fragment amplification 1 μl PCR Index Primer 10 μl 5X HF Phusion Buffer 1 μl 10 mM dNTP 0.5 μl Phusion DNA Polymerase up to 50 μl Sterile Double-Distilled Water 50 μl Total Volume Amplify using the following PCR protocol: list-behavior=enumerated prefix-word= mark-type=lower-alpha max-label-size=0 30 sec at 98°C 18 cycles of: list-behavior=enumerated prefix-word= mark-type=lower-roman max-label-size=0 10 sec at 98 °C 30 sec at 65 °C 30 sec at 72 °C 5 min at 72 °C Hold at 4 °C until ready to proceed with next step.

Techniques: Sequencing, Purification, Modification

Journal:

Article Title: Preparing DNA Libraries for Multiplexed Paired-End Deep Sequencing for Illumina GA Sequencers

doi: 10.1002/9780471729259.mc01e04s20

Figure Lengend Snippet: Fragmented DNA smearing patterns after ligation of adapters (Ligation Mix) and after PCR (PCR Mix). Note increase in smear intensity as well as a shift up in the size range of the smear.

Article Snippet: Materials list-behavior=simple prefix-word= mark-type=none max-label-size=0 Adapter-ligated DNA sample PCR Primer InPE1.0 (Illumina Cat. #PE400-1001) PCR Primer InPE2.0 (Illumina Cat. #PE400-1001) PCR Index primer (Illumina Cat. #PE400-1001) 5x HF Phusion buffer (NEB Cat. #F-540S) 10 mM dNTP (NEB Cat. #N0447S) Phusion DNA polymerase (NEB Cat. #F-540S) 6X gel loading dye with Gel Green (see recipe) 100 bp Marker (NEB Cat. #N3231S) 1X TBE (see recipe) 5% TBE DNA acrylamide gel (BioRad Cat. #161-1109) Sterile double-distilled water (nuclease-free) PCR thermal cycler to hold 200 μl PCR tubes with heated lid DNA acrylamide gel electrophoresis set-up QIAquick PCR Purification kit (Qiagen Cat. #28104) NanoDrop spectrophotometer (NanoDrop Model #ND100) list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Prepare the following reaction mix on ice in a new 200 μl PCR tube, making sure that the Phusion DNA polymerase is the last component to be added to the mix: list-behavior=enumerated prefix-word= mark-type=lower-alpha max-label-size=0 The PCR Index Primer used should be different for each strain DNA library constructed (e.g. strain 1 – Index Primer 1; strain 2 – Index Primer 2; strain 3 – Index Primer 3; etc) 175 ng Adapter-Ligated DNA sample 1 μl PCR Primer InPE1.0 for fragment amplification 1 μl PCR Primer InPE2.0 for fragment amplification 1 μl PCR Index Primer 10 μl 5X HF Phusion Buffer 1 μl 10 mM dNTP 0.5 μl Phusion DNA Polymerase up to 50 μl Sterile Double-Distilled Water 50 μl Total Volume Amplify using the following PCR protocol: list-behavior=enumerated prefix-word= mark-type=lower-alpha max-label-size=0 30 sec at 98°C 18 cycles of: list-behavior=enumerated prefix-word= mark-type=lower-roman max-label-size=0 10 sec at 98 °C 30 sec at 65 °C 30 sec at 72 °C 5 min at 72 °C Hold at 4 °C until ready to proceed with next step.

Techniques: Ligation