Rui:LabNotes/SingleCell/2011-1-26: Difference between revisions

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  *Ligation reaction sets up to 10ul (1ul:1ul PCR:p2.1; 5ul buffer; 0.5ul ligase; 5ul water), 5min at R.T.
  *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)
  *4 tubes (NTC-ES, NTC-EB, 10pg-EB, 500pg-EB)
==1st strand cDNA synthesis==
*RT Hues54, Hues6-ES, Hues6-EB 1st cDNA
*3ul of original total RNA samples
*0.5ul of P1 primer (100uM)
*Following Invitrogen 1st strand cDNA synthesis kit protocol

Revision as of 00:25, 27 January 2011

Trouble shooting for PCR

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
Aim 1: check contamination in Rui water; 10pg RNA sample
Aim 2: check primer hetero-dimer
SYBR(-) and Taq(-) are negative control


File:Tube1-4.jpg
File:Tube5-8.jpg
File:Tube9-10.jpg


Thoughts:
*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

*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)


1st strand cDNA synthesis

*RT Hues54, Hues6-ES, Hues6-EB 1st cDNA
*3ul of original total RNA samples
*0.5ul of P1 primer (100uM)
*Following Invitrogen 1st strand cDNA synthesis kit protocol