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
  1. 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
  1. Purified by AMpure beads, eluted in 36ul water, as check in 1.27.11 lab note [4]
  2. Concentration by Nanodrop, as shown in 1.27.11 lab note [5]