Team:Goettingen/NoteBook w7

Transformation from 15.7.13

-          Amp-plates:

negative control = negative/no clones

transformation of RBS-GFP-Terminator C12 plasmid: many clones on 50 µl and on rest plate

-          Cm-plates

Negative control = negative

Clones on all other plates including w/o insert control (forgot gel extraction of pSB1C3 vector à terminator insert is still in reaction mix; since we didn’t dephosphorylate the vector with AP, there will be false positives corresponding to plasmid part 7!); smaller and larger clones on all plates; further incubation over day since they were too small to be picked in the morning…

 

Colony PCR for RBS-GFP-Terminator C12 re-trafo clones 1 to 10 (RT-C1 to RT-C10)

-          According to protocol from 10.7.13

-          16x MasterMix

-          Clones 1 – 10 picked, streaked out on LB^Amp-plate and used for inoculation of 25 µl reactions (blue tubes)

-          Controls: 1 µl of 1:10 dilution of plasmids part 7, part 8 GFP-Terminator C1 and RBS-GFP-Terminator C12 (yellow tubes)

Concentration of dialysed protein samples

-       Put E1 samples from dialysis in vivaspin column

-       Spin column for 20min at 4000xg

-       Add 4ml of E2 to vivaspin and centrifuge for 20min at 4000xg

-       Resuspend and repeat centrifugation and resuspension

 

 

Determination of final protein concentration via Bradford test

-       Add 1ml of Bradford solution (1:5 dilution = 200ml Bradford + 800ml H₂O) to 5µl of 1:20 diluted protein solution

-       Incubation for 5min at RT

-       Measure extinction at 595nm

-       Calculation: c= OD₅₉₅ / Vx0,0536

Eluate	Absorption (nm)	C [µg/ml]
Sample 1	0,121	0,49
Sample 2	0,123	1,49
Total: 9,35 mgml

 

SDS Page of dialysis and elution samples

-       Mix 15µl of samples  with 5µl Pab and 5µl buffer W

-       Incubation for 10min at 93°C

-       Mix each sample with 4µl protein marker

-       Load whole volume on 15% SDS-gel

-       Run for 5min at 90V and 1h at 120V

D1 | D2 | D3 | M | E1 | E2 |

 

Analysis of the RD samples from 12.7.

Test run of plasmids purified on 12.7.13, vector and inserts on agarose gel

• part 7 plasmid, purified on 12.7.13
• pSB1C3 vector from part 7, purified on 11.7.13
• plasmid RBS-GFP-Terminator C12
• plasmid RBS-GFP-Terminator C12 test restriction with EcoRI
• inserts of DarR/riboswitches after 2nd digest with EcoRI
• 1% agarose-1xTAE-gel
• 3 µl 2 log ladder loaded on both sides of gel as a marker

Samples:

Component	Part 7 plasmid (179.3 ng/µl)	pSB1C3 (2nd elution, 3.4 ng/µl)	plasmid RBS-GFP-Terminator C12 (200.8 ng/µl)	plasmid RBS-GFP-Terminator C12 R.D.	DarR purified PCR product (4 µl primer; reaction from 24.6.13; 15.2 ng/µl)	DarR insert
DNA	0.5 µl	4 µl	0.5 µl	5 µl	2 µl	3 µl
dH2O	3.5 µl	-	3.5 µl	-	2 µl	1 µl
5xLD	1 µl	1 µl	1 µl	-(sample in green FD buffer	1 µl	1 µl

Component	Riboswitch iGEM_40/41 Purified PCR product (45.7 ng/µl)	Riboswitch iGEM_40/41 insert	Riboswitch iGEM_41/42 Purified PCR product (36.8 ng/µl)	Riboswitch iGEM_41/42 insert	Riboswitch iGEM_40/43 Purified PCR product (47.7 ng/µl)	Riboswitch iGEM_40/43 insert	Riboswitch iGEM_42/43 Purified PCR product (35.7 ng/µl)	Riboswitch iGEM_42/43 insert
DNA	1 µl	3 µl	-	3 µl	1 µl	3 µl	-	3 µl
dH2O	3 µl	1 µl	4 µl	1 µl	3 µl	1 µl	4 µl	1 µl
5xLD	1 µl	1 µl	1 µl	1 µl	1 µl	1 µl	1 µl	1 µl

(for Riboswitch iGEM_41/42 and _42/43, there were too small amounts of PCR product left -> control was not possible…)

• Run at 100 V
• EtBr staining + destaining in water
• UV detection

Purification of DarR and Riboswitch inserts

• Qiagen PCR purification kit
• 500 µl PB used for each reaction
• 2x elution in pre-warmed HPLC water (25 µl)
• NanoDrop concentration measurements (for some samples, the concentration was measured twice, since first values for 1st and 2nd elution were a little strange… -> approximate average concentration can be used for further calculations)

1st elution

Sample	Concentration (ng/µl)	Average concentration	A260/A280	A260/A230
Riboswitch iGEM_40/41 insert	7.5		1.74	1.01
Riboswitch iGEM_40/43 insert	5.5		1.18	1.09
Riboswitch iGEM_42/41 insert	2.3	3	1.89	0.86
	4.6		1.38	0.76
Riboswitch iGEM_42/43 insert	3.0	3	2.18	0.76
	3.1		1.09	0.90
DarR insert	1.7	2	3.34	0.71
	2.4		1.06	0.62

2nd elution

Sample	Concentration (ng/µl)	Average concentration	A260/A280	A260/A230
Riboswitch iGEM_40/41 insert	5.2		1.30	0.56
Riboswitch iGEM_40/43 insert	1.5		0.99	0.36
Riboswitch iGEM_42/41 insert	2.6	3	1.38	0.52
	3.6		1.18	0.62
Riboswitch iGEM_42/43 insert	3.9	4	1.14	0.63
	4.2		1.07	0.61
DarR insert	2.9		1.04	0.52