Demonstrate - Gold
Friday, 09/29/17
-colony PCR for LacI-dCas9 in pSB1C3
-4 clones, all negative
-amplification of P1 and IAA-fusion for assembly of P4:
Q40/Q41:
- amplification of P1 creating new overhangs for Gibson assembly --> 3,4 kb
Q42/Q43:
- amplificaton of IAA-fusion creating new overhangs for Gibson assembly --> 4 kb
Q44/Q45:
- amplification of IAA-fusion creating overhangs for restriction enzymes to cut more efficiently
-made 50uL reactions and ran test gel (left 4 bands are colony PCR then 40/42/44)
-only 42 has correct band
-gel extraction: 33,7 ng/ul
-named Eppi "IAA-fusion for Gibson"
-gradient PCR for 40 and 44:
-10 µL reaction and gel run
-40/1,4,5,6 and 44/1 were positive --> a PCR-clean-up has to be done
-repeat PCR from 27.07.2017:
A2: 1µl Template 59°C
A4: 1µl Template 63,3°C
A5: 1µl Template 66,5°C
B4 0,1µl Template 63,3°C
(A2, A4, A5 and B5 are the names of the original well names of the gradient PCR)
-just A 5 was positive -> a gel clean up has to be done the next day
-colony PCR for LacI-dCas9 in pSB1C3
-4 clones, all negative
-amplification of P1 and IAA-fusion for assembly of P4:
Q40/Q41:
- amplification of P1 creating new overhangs for Gibson assembly --> 3,4 kb
Q42/Q43:
- amplificaton of IAA-fusion creating new overhangs for Gibson assembly --> 4 kb
Q44/Q45:
- amplification of IAA-fusion creating overhangs for restriction enzymes to cut more efficiently
-made 50uL reactions and ran test gel (left 4 bands are colony PCR then 40/42/44)
-only 42 has correct band
-gel extraction: 33,7 ng/ul
-named Eppi "IAA-fusion for Gibson"
-gradient PCR for 40 and 44:
well | temperature in °C |
---|---|
1 | 52 |
4 | 55 |
5 | 57 |
6 | 59 |
7 | 61 |
8 | 63 |
9 | 65 |
12 | 68 |
-10 µL reaction and gel run
-40/1,4,5,6 and 44/1 were positive --> a PCR-clean-up has to be done
-repeat PCR from 27.07.2017:
A2: 1µl Template 59°C
A4: 1µl Template 63,3°C
A5: 1µl Template 66,5°C
B4 0,1µl Template 63,3°C
(A2, A4, A5 and B5 are the names of the original well names of the gradient PCR)
-just A 5 was positive -> a gel clean up has to be done the next day
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