kaitlynrm 19:28 on February 12  

Kaitlyn’s notebook: testing new primers on geoduck hemolymph RNA

I made cDNA from previously isolated RNA. I included a pooled sample that I used to test the primers I recently designed via qPCR.

Reverse Transcriptase

RT was performed using 100ng of each sample with M-MLV Reverse Transcriptase from Promega according to the Roberts Lab SOP. All reagents were doubled to produce twice the amount of cDNA. The pooled sample was quadrupled. A 1:1000 dilution of primer was made to increase pipetting volume, and a 1:10 dilution was made for the pooled sample. Calculations can be found here.

  • Pooled sample contains 61, 55F, 43B, and 19

Samples are stored in the -20C fridge in 209 in 20190107 Kaitlyn’s Box.

qPCR

Primers were tested based on the Roberts lab qPCR SOP. The primers can be found here or they can also be found in the PrimerDatabase.

There was more pooled sample loss than I expected from RT. I originally intended to use 4ul of pooled sample per primer. Instead I could only use 1ul per sample, and I spiked in 1ul of cDNA from pre-existing cDNA so that I had enough master mix avaliable for each sample.

Results

meltcurvemeltpeakqpcr2

Well Fluor Target Content Sample Cq
A01 SYBR TIF3s6B Unkn-01 Pooled 18.2612
A02 SYBR TIF3s6B Unkn-01 Pooled 18.17076
A03 SYBR NFIP Unkn-02 Pooled 27.42391
A04 SYBR NFIP Unkn-02 Pooled 26.68092
A05 SYBR APLP Unkn-03 Pooled 27.29234
A06 SYBR APLP Unkn-03 Pooled 27.19591
A07 SYBR TIF3s10 Unkn-04 Pooled 37.34317
A08 SYBR TIF3s10 Unkn-04 Pooled 33.8457
B01 SYBR ECHD3 Unkn-05 Pooled 32.67729
B02 SYBR ECHD3 Unkn-05 Pooled 33.01202
B03 SYBR TIF3sF Unkn-06 Pooled 21.97772
B04 SYBR TIF3sF Unkn-06 Pooled 22.08964
B05 SYBR TIF3s12 Unkn-07 Pooled 32.0917
B06 SYBR TIF3s12 Unkn-07 Pooled 32.74342
B07 SYBR FEN1 Unkn-08 Pooled 34.8731
B08 SYBR FEN1 Unkn-08 Pooled NaN
C01 SYBR SPTN1 Unkn-09 Pooled 27.68292
C02 SYBR SPTN1 Unkn-09 Pooled 27.16297
C03 SYBR NSF Unkn-10 Pooled 34.57384
C04 SYBR NSF Unkn-10 Pooled 37.76697
C05 SYBR TIF3s7 Unkn-11 Pooled 34.7186
C06 SYBR TIF3s7 Unkn-11 Pooled 35.08897
C07 SYBR TIF3s8-2 Unkn-12 Pooled 22.06597
C08 SYBR TIF3s8-2 Unkn-12 Pooled 21.92805
D01 SYBR TIF3s8-1 Unkn-13 Pooled 37.13184
D02 SYBR TIF3s8-1 Unkn-13 Pooled 35.01336
D03 SYBR TIF3s4a Unkn-14 Pooled NaN
D04 SYBR TIF3s4a Unkn-14 Pooled NaN
D05 SYBR GLYG Unkn-15 Pooled 37.49771
D06 SYBR GLYG Unkn-15 Pooled 35.38684
D07 SYBR RPL5 Unkn-16 Pooled 17.46911
D08 SYBR RPL5 Unkn-16 Pooled 19.57397
E01 SYBR GSK3B Unkn-17 Pooled 19.45339
E02 SYBR GSK3B Unkn-17 Pooled 18.07122

One target primer, FEN1, only worked on one sample which may have been a technical error so it may be worth re-running. TIF3s4a, one of 9 regulatory primers, did not work on either sample, but all the others worked great!

Next…

  • Choose 1-2 regulatory primers
  • Retest FEN1, target, primer
  • Assess known samples