investigated cDNA incubation time and pcr product quality 05-16-2014d1004
2014-08-29investigated cDNA incubation time and pcr product quality 05-16-2014d1004
Were PCRs better from cDNA from 30 minute incubation compared with cDNA from 15 min incubation?
Location of data
\\biofs.biodesign.asu.edu\CIM\Research\labdata\Felicia Craciunescu\cancer vaccine project 2013\frameshift pcr screen\5-16-14
and
C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2014\05-16-2014d1005\investigation of cdna incubation time and pcr product quality
Notes
15 min incubation samples
1099 15
944 15
399 15
295 15
1422 15
188 15
119 15
30 min incubation samples
328N1
350R1
411R1
637R1
1185R1
1228R1
cDNA Sample Incubation Time
1099 15
944 15
399 15
295 15
1422 15
188 15
119 15
328N1 30
350R1 30
411R1 30
637R1 30
1185R1 30
1228R1 30
tube sample primer Incubation time for cDNA
1 1228 GF60 30
2 1185 GF60 30
3 328 GF60 30
4 1165 GF38 ?
5 1099 GF38 15
6 944 GF38 15
7 350 GF79 30
8 1228 GF79 30
9 399 GF79 15
10 295 GF148 15
11 1422 GF148 15
12 188 GF109 15
13 350 GF109 30
14 411 GF109 30
15 119 GF021 15
16 637 GF021 30
Evaluation of cDNA incubation time on PCR Screen 5-16-14 spreadsheet
"\\biofs.biodesign.asu.edu\CIM\Research\labdata\Felicia Craciunescu\cancer vaccine project 2013\frameshift pcr screen\5-16-14\Evaluation of cDNA incubation time on PCR Screen 5-16-14.xlsx"
http://www.thermoscientificbio.com/uploadedFiles/Resources/k1641-product-information.pdf
Earlier we were wondering if the cDNA produced with a 15 min incubation time gave us worse PCR products. In this case, the opposite seems to be true.
Overall cDNA that was made with the 15 min incubation time yields better PCR products than cDNA that was made with the 30 min incubation time. This observation may be due to the nature of the PCR sequences rather than the incubation time, but these results are something to consider. Note that the original protocol states that the incubation time should be extended to 30 min if there is more than 1 ug RNA. The official dynazyme protocol is located here: http://www.thermoscientificbio.com/uploadedFiles/Resources/k1641-product-information.pdf
Therefore, I suppose we should just continue to follow the original protocol as we did the first time.
Anyway, I look forward to trying out the nested PCR!