Team:Potsdam/laborbuch/lowcopy

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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:

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

Monday, 10/02/17

-gel-clean-up LacI-dCas9 (A5) and PCR-clean-up 40/1 and 44/1 (40/4,5,6 were found in the waste, 40/4 was empty, because open cap, but 40/5,6 were also clean-up by PCR clean-up and purifien over one column but separated from 40/1 to not possibly destroy the one safe sample)
-concentration: 40/1: 19,7 ng/µl
-40/5,6: 31 ng/µl (but probably waste/just denatured DNA or something else)
-44/1: 30,6 ng/µl
-LacI-dCas9: 25,9 ng/µl
-Eppi-names: "IAA-Fusion for Restriction" name of 44/1 (is from PCR with Q44 and Q45)
-"P1 + overhangs 1" name of 40/1 and "P1 + overhangs 5,6" name of 40/5,6 (is from PCR with Q40 and Q41)
-Restriction of IAA-Fusion (is from PCR with Q44 and Q45):
10,2µl IAA-Fusion (19,7ng/µl) --> 200ng
1,5µl Eco-RI-HF
1,5µl PstI
1,5µl NEB2.1
0,3µl water
-total volume: 15µl
-final concentration of IAA-Fusion: 13,3ng/µl
-was incubated 1h 35min at 37°C
-Ligation of digested IAA-Fusion in pSB4A5 = P1
-176,7ng Insert = 13,25µl Iaa-Fusion digested (13,3 ng/µl)
-50ng backbone = 2,5µl pSB4A5 digested (with EcoRI and PstI) (19.8ng/µl)
-1,25µl water
-2µl Ligase-buffer
-1µl Ligase
-total volume: 20µl
-90 min at room temperature
-for ligation an insert-backbone-ratio of 3:1 was choosen
-IAA-Fusion (Insert): 4000bp
-pSB4A5 (backbone): 3395bp
-(4000bp/3395bp) * 50ng * 3 = 176,7ng = m(Insert)
-Restriction of LacI-dCas9 for iGEM-HQ of Part P5:
-digestion of 400ng LacI-dCas9 = 15,4µl LacI-dCas9
-15,4µl LacI-dCas9 (25,9ng/µl)
-2µl NEB2.1
-0,5µl EcoRI
-0,5µl PstI
-1,6µl H2O
-total volume: 20µl
-final concentration LacI-dCas9: 20ng/µl
-was incubated for 1h15min at 37°C
-Ligation of digested LacI-dCas9 in pSB1C3 for iGEM-HQ:
-Ligation was sheduled in a 1:1 Insert:backbone-ratio, because the insert is much bigger than the backbone and a very high concentration of insert would be needed to get this ratio in a 20µl reaction. Such a high concentration we don't have. But the concentration was this time at least high enough for a 1:1-ratio with 50ng of backbone.
-size Insert: 5700bp
-size backbone: 2000bp
-(5700bp/2000bp)*50ng*1 = 142,5ng
-142,5ng LacI-dCas9 (20ng/µl) = 7,1µl LacI-dCas9
-7,1µl LacI-dCas9 digested (20ng/µl; 142,5ng)
-4µl pSB1C3 digested (12,5ng/µl; 50ng)
-2µl Ligase-buffer
-1µl Ligase
-5,9µl H2O
-total volume: 20µl
-incubated 90 min at room temperature
-Gibbson-Assembly of IAA-Fusion with P1=pSB4A5:
-2,5µl pSB4A5 linearised (19,7ng/µl; 50ng; P1 with new overhangs from today PCR-clean-up, Eppi-name: P1 +overhangs 1") -> we used 40/1, because even if 5/6 has a higher concentration, 5/6 was the whole weekend not cooled in the waste and we can not be sure, that what we measured as concentration is really our DNA-fragment of interest, if this Gibbson-Assembly should not work we can try with 5/6 or better we check 5/6 on a gel before
-3,5µl IAA-Fusion for Gibbson (33,7ng/µl; 116,6ng; from Friday 29.09.)
-4µl water
-10µl Gibbson-Master-Mix
-total volume: 20µl
-IAA-Fusion + pSB4A5 reaction for 19 min at 50°C incubated
-Transformation of
-IAA-Fusion in pSB4A5 (Restriction-Ligation)
-IAA-Fusion in pSB4A5 (Gibbson-Assembly)
-LacI-dCas9 in pSB1C3 (Restriction-Ligation)
-in chemo-competent E.coli-cells (followed transformation protocol)
-Inactivation of ligase of the both ligase reactions after transformation for 10min at 65°C
-eppis with names "Ligation IAA-Fusion in pSB4A5" and "Ligation LacI-dCas9 in pSB1C3" stored at -20°C
-Gibbson-Assembly with eppi name: "Gibbson-Assembly IAA-Fusion in pSB4A5" stored at -20°C
-plating of the cells after 1h incubation at 37°C: 200µl and pellet, respectively

Tuesday, 10/03/17

-colony-PCR of IAA-fusion in psB4A5 (Gibson-Assembly) (primer: IG_C_Q17 and IG_C_Q23)
-no colony-PCR of restriction-ligation, because no colonies
-colony-PCR of LacI-dCas9 in psb1C3 (restriction-ligation) (primer: IG_C_Q17 and IG_C_Q20)
-for all colony-PCR following PCR-program:

-initial denaturation: 1 min 95 °C

-denaturation: 15 sec 95 °C

-annealing: 15 sec 52 °C

-extension: 30 sec 72 °C

-final extension: 5 min 72 °C

-40 cycles

-run 10 µl PCR-reaction (each) on 0,7 % agarose gel

-result: - LacI-dCas9: all negative

- IAA-fusion in psB4A5: 1 and 4 possible postiv

=> ideas: dephosphorylate psb1C3 before ligation to prevent selfligation, because PstI in NEB 2.1- Buffer 75 % activity, so possible plasmid only cut by EcoRI


-minpreps of colonie 1 and 4 (IAA-fusion in pSB4A5)