First Strand cDNA Synthesis (AB Kit) - Revision history

Snyderds: adjusted quantity of RNA added

2019-07-24T15:16:23Z

adjusted quantity of RNA added

← Older revision		Revision as of 15:16, 24 July 2019
Line 36:		Line 36:

	* Add 10 uL Master mix per well into tube or plate		* Add 10 uL Master mix per well into tube or plate
	* Add ~~0.1-~~10 ug RNA in a final volume of 10 uL to each well		* Add 10 ug RNA in a final volume of 20 uL to each well. Since results will be normalized in the end, standardizing RNA quantities at this step is unnecessary. Total volume can be adjusted down if RNA is in short supply.
	* Run the PCR Program '''AB Reaction Protocol'''. Takes ~2h:		* Run the PCR Program '''AB Reaction Protocol'''. Takes ~2h:
	** 10 min at 25C		** 10 min at 25C
	** 120 min at 37C		** 120 min at 37C
	** 5 min at 85C		** 5 min at 85C

Reddj at 15:33, 8 May 2017

2017-05-08T15:33:59Z

← Older revision		Revision as of 15:33, 8 May 2017
Line 37:		Line 37:
	* Add 10 uL Master mix per well into tube or plate		* Add 10 uL Master mix per well into tube or plate
	* Add 0.1-10 ug RNA in a final volume of 10 uL to each well		* Add 0.1-10 ug RNA in a final volume of 10 uL to each well
	* Run the PCR Program '''AB Reaction'''. Takes ~2h:		* Run the PCR Program '''AB Reaction Protocol'''. Takes ~2h:
	** 10 min at 25C		** 10 min at 25C
	** 120 min at 37C		** 120 min at 37C
	** 5 min at 85C		** 5 min at 85C

Davebrid: Added AB Reaction protocol

2015-12-08T17:58:57Z

Added AB Reaction protocol

← Older revision		Revision as of 17:58, 8 December 2015
Line 37:		Line 37:
	* Add 10 uL Master mix per well into tube or plate		* Add 10 uL Master mix per well into tube or plate
	* Add 0.1-10 ug RNA in a final volume of 10 uL to each well		* Add 0.1-10 ug RNA in a final volume of 10 uL to each well
	* Run the PCR Program '''AB Reaction'''. Takes ~2h		* Run the PCR Program '''AB Reaction'''. Takes ~2h:
			** 10 min at 25C
			** 120 min at 37C
			** 5 min at 85C

Davebrid: added categories

2012-06-01T16:45:28Z

added categories

← Older revision		Revision as of 16:45, 1 June 2012
Line 1:		Line 1:
			[[Category:qPCR]]
			[[Category:RNA]]
			[[Category:Transcription]]
			[[Category:mRNA]]
			[[Category:Mouse Work]]

	==Materials==		==Materials==
	* Purified RNA		* Purified RNA

Davebrid: Wrote protocol for AB Kit

2012-06-01T16:27:17Z

Wrote protocol for AB Kit

New page

==Materials==
* Purified RNA
* High Capacity cDNA Reverse Transcription Kit without RNAse inhibitor (AB 4368813 for 1000 reactions, see http://products.invitrogen.com/ivgn/product/4368813)

==Protocol==
* Generate Master Mix Containing:

{| border="1"
|+ Master Mix Components
! scope="col" | Component
! scope="col" | Per Reaction
! scope="col" | Per 10 Reactions
|-
! scope="row" | 10X RT Buffer
| 2 || 20
|-
! scope="row" | 25X dNTP Mix (100 mM)
| 0.8 || 8
|-
! scope="row" | 10X RT Random Primers
| 2 || 20
|-
! scope="row" | MultiScribe Reverse Transcriptase
| 1 || 10
|-
! scope="row" | Nuclease Free Water
| 4.2 || 42
|-
|}

* Add 10 uL Master mix per well into tube or plate
* Add 0.1-10 ug RNA in a final volume of 10 uL to each well
* Run the PCR Program '''AB Reaction'''. Takes ~2h