Wednesday, August 19, 2015

8 18 2015 18s/28s primer check qPCR

While looking for new normalizing primers to add to the EF1d and actin that I already have I've developed primers for 18s and 28s. The 18s are based on sequences for Ostrea conchaphila. The 28s primer is developed from the 28s sequence from the O.lurida Transcriptome version 3. I tested these primers on the mechanical stress samples in a 1:9 dilution.

170228s_3_FWDTAAGGCCAGTGTGGGAGAGAJH8/12/2015205559.88O.lurida"28S ribosomal protein S5, mitochondrial (MRP-S5) (S5mt)"Q5REJ1
170128s_3_REVCGCCTCACTTTTTGTGCCTCJH8/12/2015205560.04O.lurida"28S ribosomal protein S5, mitochondrial (MRP-S5) (S5mt)"Q5REJ1
170018s_2_FWDCTCGATTCCCTTTCCCCCACJH8/12/2015206060.11O.conchaphila18s Ribosomal RNAEF035117.1
169918s_2_REVGCCCGATCTCTTTCCACCTCJH8/12/2015206060.18O.conchaphila18s Ribosomal RNAEF035117.1
169818s_1_FWDTTCCTTTTCCCCCACTCAGCJH8/12/2015205559.89O.conchaphila18s Ribosomal RNAEF035118.1
169718s_1_REVCGCTTCACTCGCCGTTACTAJH8/12/2015205560.18O.conchaphila18s Ribosomal RNAEF035118.1

Reagent Table:
VolumeReactions X10
Ssofast Evagreen MM 10100
FWD Primer0.55
REV Primer0.55
PCR H2O550
1:9 cDNA4
  1. Added reagents from greatest to least volume
  2. Vortexed
  3. Centrifuged briefly
  4. Pipetted 16 ul Master Mix to each tube
  5. Pipetted 4 ul of 1:9 cDNA each column using a channel pipetter
  6. Centrifuged plate at 2000 rpm for 1 minute
  7. Ran Program Below

Initiation95 C10 min
Elongation95 C30 sec
60 C1 min
72 C30 sec
Repeat Elongation 39 times
Termination95 C1 min
55 C1 sec
Melt Curve Manual ramp 0.2C per sec Read 0.5 C55 - 95 C30 sec
21 C10 min
Table Layout:
DNased 42215 HM1 1DNased 42215 NM1 1DNased 42215 SM1 118s_1_NTC18s_1_NTC
DNased 42215 HM1 2DNased 42215 NM1 2DNased 42215 SM1 218s_2_NTC18s_2_NTC
DNased 42215 HM1 3DNased 42215 NM1 3DNased 42215 SM1 328s_3_NTC28s_3_NTC
DNased 42215 HM1 4DNased 42215 NM1 4DNased 42215 SM1 4
DNased 42215 HM1 5DNased 42215 NM1 5DNased 42215 SM1 5
DNased 42215 HM1 6DNased 42215 NM1 6DNased 42215 SM1 6
DNased 42215 HM1 7DNased 42215 NM1 7DNased 42215 SM1 7
DNased 42215 HM1 8DNased 42215 NM1 8DNased 42215 SM1 8

The 18s primers each have issues. The first has seemingly random amplification and poor melt curves. The second seems to autoamplify and has NTCs with amplification equal to the samples. 
The 28s primer looks great. It has strong amplification with only primer dimers appearing in the NTCs. I think this should be used for the next normalizing primers. 

No comments:

Post a Comment