Team:Heidelberg/Templates/Indigoidine week17

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Contents

Preparation for T-Domain exchange

Fragment Amplification

Table 12.x PCR for T/TE-Domain exchange with CPEC. Phusion Flash HF
template: 0.5 ul from pRB19 miniprep, Primer KH3/4
template: 0.5 ul from pRB19 miniprep, Primer KHRB21/KH4
cyclestemperature (°C)time (s)
19810
35981
605
72100
172300
112-

No band at 6400 bp. Second try with less stringent conditions

Table 12.x PCR #2 KH3/4 from pRB19, Phusion Flash HF
template: 0.5 ul from pRB19 miniprep, Primer KH3/4
template: 0.5 ul from pRB19 miniprep, Primer KHRB21/KH4
cyclestemperature (°C)time (s)
19810
10 981
62 ? 0.55
72100
25981
575
72100
172300
112-

Still nothing; maybe the MiniPrep didn't work so fine. We will screen pRB19-transformed cells on pRB19. We want to get a first impression on whether the Domain-exchange works, so we'll use pRB14 instead.

Table 12.x PCR #2 KH3/4 from pRB14, Phusion Flash HF
template: 0.8 ug from pRB14 miniprep, Primer KH3/4
template: 0.8 ug from pRB14 miniprep, Primer KHRB21/KH4
cyclestemperature (°C)time (s)
19810
10 981
62 ? 0.55
72120
25981
575
72120
172300
112-

PCR worked but with very low yield. We try to amplify from pRB19-transformed cells instead of the miniprep.

Table 12.x PCR #2 KH3/4 from pRB19, Phusion Flash HF
template: 1 ul from pRB19-transformed cells, Primer KH3/4
template: 1 ul from pRB19-transformed cells, Primer KHRB21/KH4
cyclestemperature (°C)time (s)
19810
30 981
57-60-685
72120
172300
112-


25 ul Phusion HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

Table 12.x Table 12.x PCR TTE-domains
TTE-Domains in BioRAD cycler 2
bpsA-TTE KH7/RB72 1 ng pMM64 miniprep
entF-TTE RB53/73 colony MG1655
tycC6-TTE RB57/74 1 ul B. para overnight culture
delH5-TTE RB61/75 colony D. aci SPH-1
cyclestemperature (°C)time (s)
198120
409810
6520
7230
172120
112-

bpsA-TTE and tycC6-TTE worked, the other ones will be amplified using less stringent conditions

25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

Table 12.x Table 12.x PCR TTE-domains
TTE-Domains in BioRAD T100
entF-TTE RB53/73 colony MG1655
delH5-TTE RB61/75 colony D. aci SPH-1
cyclestemperature (°C)time (s)
198120
10 981
62 ? 0.55
7220
259810
605
7220
17260
112-

delH5-TTE worked, though with low yield. entF-TTE will be amplified in a third PCR.

25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

Table 12.x Table 12.x PCR TTE-domains
TTE-Domains in BioRAD T100
entF-TTE RB53/73 colony MG1655
cyclestemperature (°C)time (s)
198120
10 981
60 ? 0.55
7220
259810
555
7220
17260
112-

entF-TTE didn't work. We will postpone this PCR.

T-Domain exchange

CPEC Assembly and Transformation

PlasmidFragment 1Molarity [nM]Volume in ulFragment 2Molarity [nM]Volume in MMCPEC Master Mix total [ul]
pRB14-T1pRB14d(ccdB)3.12.7 indC-T1457.60.36
pRB14-T2pRB14d(ccdB)3.1 2.7 bpsA-T1506.10.36
pRB14-T4pRB14d(ccdB)3.1 2.7 tycA1-T1395.50.36
pRB14-T5pRB14d(ccdB)3.1 2.7 tycC6-T1504.50.36
pRB14-T6pRB14d(ccdB)3.1 2.7 delH4-T1325.80.36
pRB14-T7pRB14d(ccdB)3.1 2.7 delH5-T1230.30.36
pRB14-TTE1pRB14d(ccdB-TE)33.5 3bpsA-TTE140.8210
pRB14-TTE3pRB14d(ccdB-TE)33.5 1.5tycC6-TTE31.83.510
pRB14-TTE4pRB14d(ccdB-TE)33.5 1.5delH5-TTE34.73.510


Table 13.x CPEC Assembly pRB14-Tx/TTEx
BioRAD T100
CyclesTemperature [°C]Time [s]
19810
8981
535
72120
172300
112-

Transformation in TOP10 according to standard protocol. Incubation for 30 hours at 37 °C and 10 hours

on the bench.


BioBricks

Fragment Preparation

25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

Table 12.x PCR for pRB22
miniprep of pRB22 with RB68/69
cyclestemperature (°C)time (s)
19810
35981
605
72100
172300
112-

PCR worked well. To make sure that we don't transform template plasmid pRB21, we digest the pRB22d(EcoRI-SpeI) fragment Gel Extraction together with pRB22d(SpeI-EcoRI) with DpnI for four hours at 37 °C.

Table 13.x DpnI-digest for pRB22
37 °C; 4 hours
ComponentConcentrationVolume [ul]
pRB22d(EcoRI-SpeI)32.3 nM11.0
pRB22d(SpeI-EcoRi)839.4 nM2.4
CutSmart buffer10x1.6
NEB DpnI1

The Digest was purified using QiaGen Nucleotide Removal Kit. Unfortunately I dropped 2/3 onto the table... The final DNA concentration was 47.5 ng/ ul according to NanoVue. Standard CPEC assembly was performed in the old broken BioRAD cycler which didn't cool down to 53 °C but only to 58 °C for the annealing step. Hopefully it worked. TOP10 cells were transformed with 5 ul of CPEC master mix.


Transformation was successful. Ten colonies were screened using primers VF2 and KH6 in a 20 ul iTaq Master Mix according to standard protocol (50 °C).


Probes number x to x were purified using Qiaquick PCR purification kit and digested with NEB DpnI, EcoRI-HF and SpeI-HF over night to see whether pRB22 is really free of those restriction sites. The result was put on an AgaroseGel.

Lanes 1 to 4 show a slight smear beneath 200 bp. Lane 5 (probe 10) doesn't show that smear and the

specific band runs maybe 300 bp higher. So maybe the first four lanes contained restriction sites and

the last one didn't. We put probes 1 and 10 in 40 ml culture for midiprep. The midiprep will then be

digested as well so that we get certainity about the restriction sites. In parallel we did a colony PCR from probe 10 with KH3/4 using Phusion Flash HF to already have our

fragment for pRB23 for the T-Domain-exchange.

Table 13.x PCR for pRB23
miniprep of pRB23 with RB68/69
cyclestemperature (°C)time (s)
198120
10981
TD 625
72140
25981
575
72120
172300
112-