investigated cDNA incubation time and pcr product quality 05-16-2014d1004

2014-08-29

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!

azim58wiki: