Rui:LabNotes/SingleCell/2011-1-26
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Trouble shooting for PCR[edit]
QPCR set-up[edit]
Aim 1: check contamination in Rui water; 10pg RNA sample Aim 2: check primer hetero-dimer
Components in tubes ---------------------------------------------------------------------------------- ID Template P1 P2 others Taq SYBR ---------------------------------------------------------------------------------- Tube1 Rui water Yes Yes Yes Yes Tube2 Kun water Yes Yes Yes Yes Tube3 Sam water Yes Yes Yes Yes Tube4 10pg RNA Yes Yes Yes Yes Tube5 Rui water Yes Yes Yes Tube6 Rui water Yes Yes Yes Tube7 Rui water P3+P4 Yes Yes Tube8 Rui water P3+p5 Yes Yes Tube9 Rui water Yes Tube10 Rui water Yes ---------------------------------------------------------------------------------- Common components: Ex Taq buffer, dNTP mix SYBR(-) and Taq(-) are negative control
QPCR curve[edit]
File:Tube1-4.jpg File:Tube5-8.jpg File:Tube9-10.jpg
Thoughts[edit]
*The amplification from contamination comes from steps above PCR *The template could possible come from primer (P1), 72bp+54bp=126bp at least *It could be the reason why 500pg amplifications have LMW bands than NTC and 10pg *Titration of Exonuclease I to remove primer 1? *If following Tang's protocol for IVT, extra primer 1 still could have problem for IVT. *Try ABi IVT kit?
Clone PCR product from PCR-based protocol[edit]
- PCR products from PCR-based protocol [1]
- Ligation and transformation
Original TA cloning kit (p2.1 w/o TOPO -- needs ligase) Use NEB quick ligation kit instead of T4 ligase provided from TA cloning kit Ligation reaction sets up to 10ul (1ul:1ul PCR:p2.1; 5ul buffer; 0.5ul ligase; 5ul water), 5min at R.T. 4 tubes (NTC-ES, NTC-EB, 10pg-EB, 500pg-EB) 15ul out of 400ul SOC media to spread on Ampicillin plate
- Colonies screen as in [2]
Positive control (cDNA) for PCR based protocol[edit]
- Continued with trouble shooting for PCR based protocol to exclude following step after free primer removal
- Use diluted cDNA (~0.01pg) as the positive control instead of 500pg RNA samples
- RT Hues54, Hues6-ES, Hues6-EB 1st cDNA with Invitrogen kit
*3ul of original total RNA samples, concentration as [3] *0.5ul of P1 primer (100uM) *Following Invitrogen 1st strand cDNA synthesis kit protocol