Arichard:Notebook/bulk150122: Difference between revisions

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* Maximum volume of HLS used to ensure hypotonicity (35 ul HLS + 35 ul G-HCl + 126 ul Ampure XP = 196 ul total in 200 ul PCR tube)
* Maximum volume of HLS used to ensure hypotonicity (35 ul HLS + 35 ul G-HCl + 126 ul Ampure XP = 196 ul total in 200 ul PCR tube)


# Lysed in 35 ul [http://genome-tech.ucsd.edu/LabNotes/index.php/Arichard:Reagents/hls 1X HLS] at RT for <5 min, followed by 35 ul of 8M guanidine HCl.
# Lyse in 35 ul [http://genome-tech.ucsd.edu/LabNotes/index.php/Arichard:Reagents/hls 1X HLS] at RT for <5 min, followed by 35 ul of 8M guanidine HCl.
# Purified with 126 ul (1.8:1 ratio) Ampure XP, washed 2X with 80% EtOH
# 126 ul (1.8:1 ratio) Ampure XP, washed 2X with 80% EtOH
# Eluted in 7 ul H2O
# Elute in 7 ul H2O
# 2 ul 5X Nextera buffer and 1 ul 1:50 Nextera v1 diluted in 50/50 TE/glycerol, 5 min at 55 C
# 2 ul 5X Nextera buffer and 1 ul 1:50 Nextera v1 diluted in 50/50 TE/glycerol, 5 min at 55 C
# 1 ul of 1:100 ProtQ (Stock is 20 mg/ml) diluted in H2O, 10 min at 50 C, 20 min at 70 C
# 1 ul of 1:100 ProtQ (Stock is 20 mg/ml) diluted in H2O, 10 min at 50 C, 20 min at 70 C

Latest revision as of 18:49, 10 February 2015

Bulk libraries January 22, 2015[edit]

  • Used Brandon's protocol for DNA purification after guanidine HCl
  • Used Blue's method of 2x Ampure XP at 0.8:1 ratio to select 200-800 bp.
  • Qiming Jin (QJ) ran most of these samples with my supervision.
  • Used SW480 2N Full sorted October 21, 2014 (tubes labeled 4 in sort box)
  • Maximum volume of HLS used to ensure hypotonicity (35 ul HLS + 35 ul G-HCl + 126 ul Ampure XP = 196 ul total in 200 ul PCR tube)
  1. Lyse in 35 ul 1X HLS at RT for <5 min, followed by 35 ul of 8M guanidine HCl.
  2. 126 ul (1.8:1 ratio) Ampure XP, washed 2X with 80% EtOH
  3. Elute in 7 ul H2O
  4. 2 ul 5X Nextera buffer and 1 ul 1:50 Nextera v1 diluted in 50/50 TE/glycerol, 5 min at 55 C
  5. 1 ul of 1:100 ProtQ (Stock is 20 mg/ml) diluted in H2O, 10 min at 50 C, 20 min at 70 C
  6. 1 ul of Exo- Klenow (Epicentre) plus 0.4 ul of 25 mM dNTPs (RepliPhi kit), 15 min at 37 C, 20 min at 75 C
  7. 0.6 ul barcoded Adapter 2 and 17 ul of PCR 1 master mix:
    1. 15 ul 2X KAPA SYBR Fast
    2. 0.6 ul Adapter 1
    3. 1.4 ul H2O
  8. PCR 1
    1. 95 C for 1 min
    2. 6 cycles:
      1. 95 C for 15 sec
      2. 62 C for 15 sec
      3. 72 C for 45 sec
  9. 70 ul of PCR 2 master mix
    1. 35 ul 2X KAPA SYBR Fast
    2. 2 ul Primer 1
    3. 2 ul Primer 2
    4. 31 ul H2O
  10. PCR 2
    1. 95 C for 1 min
    2. 7 cycles:
      1. 95 C for 15 sec
      2. 60 C for 15 sec
      3. 72 C for 45 sec
    3. 72 C for 3 min
  11. 80 ul Ampure XP (0.8:1 ratio), wash 2X with 80% EtOH
  12. Elute in 20 ul H2O

Ran first gel after first Ampure. 3 out of 20 ul of each sample loaded.

File:Bulk150122Slide1.jpg

Sample 3 (index 4) negative. Mis-loading suspected. QJ ran second gel to confirm. Another 3 ul loaded (14 ul now remaining).

File:Bulk150122Slide2.jpg

Second gel confirms that sample 3 did not amplify in PCR. Performed second Ampure XP purification:

  • 14 ul sample (20 - 3 - 3)
  • 11.2 ul beads
  • Elute in 20 ul H2O

File:Bulk150122Slide3.jpg

2X Ampure XP worked well. Smears look equivalent to PA size selection. Samples 1 and 2 submitted (QJ01 and QJ02)