Amazing Plants (Read It Yourself - Level 2) by Lorraine Horsley

By Lorraine Horsley

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5. The filtration step can be quite tedious, as the suspension is likely to clog the Miracloth. It is advisable to let the plant powder resuspend completely in NIB; thus, leave the tubes on ice for a while. If the Miracloth clogs anyway, an additional 5 ml of NIB can be added to help unclogging. If not successful, carefully remove the suspension by pipetting slowly using a 1 ml pipette tip, from which the tip was cut to widen the opening. Subsequently, filter the removed suspension through a fresh piece of Miracloth and add, if necessary, an additional 5 ml of NIB.

3 M sodium acetate. 26. Glycogen: 20 mg/ml. 27. 5 M sodium chloride. 28. 5 M EDTA. 29. 1 M Tris–HCl. 30. Tween-20. 31. Ethanol: 100 % and 70 %. 32. Agarose. 33. Ethidium bromide, be aware of the toxicity and handle correspondingly. 34. Illumina library preparation kit. 35. AMPure XP PCR purification beads (Agencourt). 36. 05 % Tween-20. 37. Binding buffer (BB) (2×): 10 mM Tris–HCl, 1 mM EDTA, 2 M NaCl. 2 Equipment 1. Vacuum pump. 2. Mortar and pestle. 3. Whatman filter paper. 4. Miracloth. 5. 15 ml and 50 ml conical tubes (Falcon tubes).

For each PCR reaction, use approx. 50 ng of the remaining Hi-C samples, which passed the prior quality control. Purify the PCR product using a standard PCR product purification kit and elute in 60 μl. 5 ml reaction tubes and set aside the remaining 10 µl of PCR product. If HindIII was used as a restriction enzyme in step 5 of this protocol, set up two restriction digest reactions, using HindIII HF and NheI HF restriction enzymes. 5 ml reaction tube. Incubate for 2 h at 37 °C. Inactivate the restriction enzymes by incubating at 65 °C (HindIII) or 80 °C (NheI) for 15 min.

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