Arichard:Notebook/2012/October

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October, 2012[edit]

10th[edit]

  • We now have Down's patient samples, Down's Neuronal and Non-neuronal (both cortex).
  • Jeff and Professor have modified the PCR protocol after tagmentation. Primers are added first, followed by 9 cycles with 58 degC annealing temp (30 ul rxn vol). Adapters are then added (to give 100 ul rxn vol) followed by 3 cycles w/58 degC anneal temp, then 5 cycles w/72 degC anneal temp. This dilution helps prevent the primers from out competing the adapters. The first 3 cycles at 58 degC with the adapters accounts for the fact that only a portion of the adapter binds to the initial template.
  • Jeff and I discussed a system for keeping track of samples and indices. From now on, each experiment will be identified by its start date, the date of MDA. This keeps a consistent date for the entire run. Below is a table showing the tube labeling for the last two runs (10-1 and 10-3):
Tube # Cap Sample and index Sample and index
1 of 5 AR 10-1 Sea1 25 Sea2 26
2 of 5 AR 10-1 Ch1 27 CtlNeu1 31
3 of 5 AR 10-1 ChDil1 29 ChDil2 30
4 of 5 AR 10-1 AlzCorNeu1 33 AlzCorNeu2 34
5 of 5 AR 10-1 DowNeu1 35 DowNeu2 36
1 of 4 AR 10-3 Ch1 29 DowNeu2 40
2 of 4 AR 10-3 ChDil1 31 ChDil2 32
3 of 4 AR 10-3 CtlNeu1 33 CtlNon1 35
4 of 4 AR 10-3 AlzCorNeu1 37 AlzCorNeu2 38
  • I need to find a better standardized way of labeling the tubes. Use + and - for neuronal and non neuronal from now on.
  • Jeff and Chris are starting a new run today (10-10). I will extract tomorrow morning.

23rd[edit]

  • The new PCR protocol has improved the number of clusters about two-fold compared to last time (two-step, but with 72 degC only during second step).
  • The number of mapped reads was very low, but Athurva and Jeff debugged the code (Bowtie was using 32 base phred score instead of 64) and we now have mapping rates of 60-90%.

24th[edit]

  • We have 4 libraries that we are trying to catalog: 10/1, 10/3, 10/10, and 10/12.
  • We are implementing a new system of data storage. Every sample will have an entry on a spreadsheet with links to each experiment page, which will contain all data including gel images, PCR data and any notes or changes to the protocol. This information will be updated in real-time for all experiments going forward.
  • The clonal rates are high for the latest libraries (70-90%). We will add BST polymerase and dNTPs to the protease reaction in order to fill in gaps left by tagmentation. This should help to prevent the loss of the priming regions added by transposase. We will continue to do Exo- treatment as well, to be safe.
  • BST has optimal activity at 65 degC, but can be used up to 70 degC. Inactivation is at 80 degC for 20 minutes.
  • We can add BST after the 15 minute protease treatment, just before the 20 minute protease inactivation at 70 degC. This will prevent the protease from destroying BST before it has had a chance to act.

25th[edit]

  • Started MDA for experiment 10/25. Sample setup is as follows (sample # in parenthesis):
Sea1 (1,2) Sea3 (5,6) Ctl+ (9,10) Ctl- (13,14)
Sea2 (3,4) Sea4 (7,8) Ctl+ (11,12) Ctl- (15,16)

26th[edit]

  • We took 2 samples from each array (1-16). Samples 1-7 and 16 will be treated with 1:10 tranposase instead of 1:50.
  • Bst will be added at the beginning of the protease treatment instead of the beginning of protease heat inactivation as we previously decided. Bst activity at 50 degC is ~35% compared to 20% at 70 degC, and this way the protease will destroy Bst and prevent it from binding to DNA and blocking subsequent Exo- treatment.
  • Only samples 1-11 have Bst.