Team:Goettingen/NoteBook w5

Transformation of Ligation with GFP-Terminator insert and part 8 backbone worked!!!

No clones on negative control, but very few clones on without insert control. à possibly again incomplete digestion of part 8 (just 20 min) à use 1 h for dig, since reaction for gel ex yesterday seemed to be more completely digested….

Stored at 4 °C

No colonies on LB^Cm plates

è Further incubation till next morning

Part 7:

15 µl part 7 plasmid DNA (1 µg)

10 µl NEB buffer 2.1

2 µl EcoRI

2 µl PstI

71 µl H₂O

 

GFP-Term:

5 µl GFP-Term C1 plasmid DNA (1 µg)

10 µl NEB buffer 2.1

2 µl XbaI

2 µl PstI

81 µl H₂O

Both reactions were split into 2x 50µl (for more thorough reaction)

Both incubated for 30 min at 37°C, then heat inactivation for 20 min at 80°C

Gel-Extraction of pSB1C3 from the restriction digest of part 7 (EcoRI+PstI) for future ligations

Gel:

Purification of DNA from gel using Qiagen PCR purification kit:

-          like Katrin 25.06.2013

Elution with 30 µl HPLC water (pre-warmed)

NanoDrop:

pSB1C3 clone	Concentration (ng/µl)	A260nm/A280nm	A260nm/A230nm
1	6.2	1.35	0.12
2	11.3	1.27	0.21
3	10.2	1.71	0.06

 

 

è DNA frozen away in one of the red boxes with today´s date on the vial.

Clonation of the RBS-GFP-Terminator Construct:

Gel-Extraction of the GFP-Terminator construct from the restriction digest (XbaI+PstI), ligation into opened RBS-Vector (Part 8) and transformation

Gel:

Purification of DNA from gel using Qiagen PCR purification kit:

-          like Katrin 25.06.2013

-          Elution with 30 µl HPLC water (pre-warmed)

NanoDrop:

GFP-Term clone	Concentration (ng/µl)	A260nm/A280nm	A260nm/A230nm
1	5.4	1.75	0.07
2	19.5	1.62	0.19
3	6.2	1.32	0.10

 

 

è DNA frozen away in one of the red boxes with today´s date on the vial.

Ligation

-Clone 2 was used for ligation to the opened RBS-Vector (also known as part 8 –see 1.7.13)

-ligation was pipetted as follows:

13.5µl HPLC H2O

2µl Ligase buffer

2.5µl RBS-Vector DNA (~20ng)

1µl GFP-Term Clone 2 DNA (~20ng)

1µl Ligase

-For the control, the GFP-Term.-Insert was substituted with HPLC H2O

-Ligation was performed at RT for 10 minutes

-Heat-inactivation was performed at 80°C for 20 minutes

Transformation

-The whole ligation was used for the transformation.

-Transformation performed according to the methods-folder

-          0.5 µl uncut plasmid + 3.5 µl dH₂O + 1 µl LD 5x

-          Ca. 40 ng of R.D. of plasmids (= 4 µl) supplied with 1 µl of LD 5x

-          Run at 100 V on 4 %-agarose-1xTAE gel; marker: log ladder (3 µl)

-          EtBr staining, destaining, UV detection

Uncut plasmids visible (between 3 and 8kb, although about 2.5kb big), linearized plasmids visible (between 8 and 10 kb…), promoter fragments not visible at all (should be 30-120bps). Will add picture later.

Discussion:

The thick gel might have unrepresentative effects on large fragments (e.g. plasmids). This thesis is supported by the unreadable marker bands between 3 and 8 kb.

The promoter fragments might just be too small to be stained properly or they are not in the constructs. Only sequencing can solve this riddle…

 

Wells used as follows:

M_Part1uncut_Part1RD_Part2uncut_Part2RD_Part3uncut_Part3RD_Part4uncut_part4RD_M

used Marker: NEB Quick-Load 2-Log DNA Ladder (0.1-10kb)

Restriction digestion of isolated plasmids from yesterday run on 1% gel (for 1.5h)

can not open scn file. Saved under Reporter team gel doc in folder with this date (gel doc 2013-07-03 15hr 29min, control = 14hr 34 min)

 

 Wells as following:

RiboA (C1,C2,C3), RiboB (C1, C2, C3), RiboC (C1), RiboD (C1, C2, C3), lin part7 (C1, C2, C3), DarR (C1, C2, C4), GFP (C1,C2,C4), pSB1C3 (C1, C2, C3)

same order of wells for control

Control:

 

Digested:

è GFP-Terminator construct could be cloned successfully (fragment of slightly > 1 kb corresponding likely to GFP (ca. 900 bp) and terminator (ca. 170 bp) visible)

è Clones derived from transformation of linearzised part 7 contain apparently plasmid with part 7 (terminator) à ca. 200 bp fragment visible after digest, corresponding presumably to terminator à clones result possibly from transformation of not linearized plasmids present after incomplete digest of part 7

è For C2 of RiboB cloning, a weak band at ca. 200 bp could be detected, that does not fit with the expected insert of 370 bp à something else got cloned…

è For transformations of ligation reactions containing linearized pSB1C3 as a vector, no transformants containing the desired inserts could be obtained à plasmid that was transformed could be one of the residual template plasmids containing Promoter-RBS-RFP-Terminator (ca. 1.5 kb): after R.D., bands at 1.5 kb and 2 kb corresponding to the expected fragments could be detected. However, band at 1.5 kb is very weak, and for uncut plasmids, a strong band at this height representing the supercoiled plasmid could be detected à it’s also possible that pSB1C3 can’t be restricted properly… anyway, linearized pSB1C3 seems not to work for cloning à cut out terminator from part 7, extract backbone (= linearized pSB1C3) and use it for cloning of Ribo- and DarR biobricks instead…

Sequencing data arrived!

àNo biobrick in the DarR operator construct!

Back-up plates

-          for all inoculated clones (except for clones containing plasmids for parts 1 – 4)

-          incubation over day at 37 °C

-          stored at 4 °C

Plasmid Mini Preparation

-          for all inoculated clones

-          kit from Macherey-Nagel

-          elution with pre-warmed 30 µl HPLC water

-          DarR biobrick clone 3: almost all lysate spilled…

-          Concentration measurement on NanoDrop

Concentrations:

è DarR biobrick clone 3 has strangely high concentration

Test restriction digest of isolated plasmids

è Cut out inserts with EcoRI and PstI (Fermentas/ThermoScientific enzymes, not distribution kit!)

è Only 3 clones tested, if there are 5 to waste less enzyme…

For 1 reaction (10 µl total volume)

0.5 µl PstI

0.5 µl EcoRI

1 µl buffer 0 (both enzymes have 100 % activity and no star activity in this buffer)

3 µl plasmid DNA mix containing ca. 200 – 300 ng plasmid (see picture of NanoDrop table above, rightmost + smallest part of table)

5 µl dH₂O

è Master Mix consisting of

12.5 µl PstI

12.5 µl EcoRI

25 µl buffer 0

125 µl dH₂O

è In total: 250 µl

è Add 7 µl to each plasmid DNA mix

Incubation for ca. 1 h at 37 °C

Freezing of samples at -20°C

 

Reactions according to manual of Distribution kit:

-          Parts 1 – 3: restriction of 1 µg plasmid DNA with SpeI and EcoRI-HF; 100 µl in total (DNA, enzyme amounts and buffer amount doubled compared to common 50 µl reaction; but distributed to 2 epis with 50 µl each à more complete reaction)

-          Part 4: restriction of ca. 250 ng plasmid DNA (8.7 µl from 29.5 ng/µl solution) with SpeI and EcoRI-HF

-          Part 8: restriction of 500 ng plasmid DNA with SpeI and PstI

-          For parts 1,2, and 3: MasterMix consisting of both enzymes and buffer and sufficient for 4 reactions was used

 

è 20 min at 37 °C digestion

è 20 min at 80 °C heat-kill

 

-          1xTAE-1%agarose gel

-          Load 3 µl of each digest (ca. 30 ng) and 1 µl of plasmid as a control, supplied with 5xDLD

-          3 µl of log ladder as marker

-          Run at 100 V

-          EtBr staining + destaining

Gel:

 

è Still, R.D. is partial à next time: try 30 min incubation

è One can conclude linearization of all plasmids, but promoter fragments are not visible…. Try higher concentrated gel and load higher amounts of DNA

è Reactions stored at – 20 °C

 

Transformation from 27.6.13

-          Colonies grew on all plates except for negative controls and those of pSB1C3 transformation

-          Colonies on without-insert-control of linearized pSB1C3 look slightly pink à remaining template from PCR transformed = pSB1C3 plasmid?

-          Colonies on without-insert-control of linearized part 7 vector might result from not digested plasmid (partial digestion, as usual….)

-          Just 1 clone for Riboswitch biobrick C…

-          Inoculation of 5 clones for each (except for Ribo C) transformation; for without-insert-controls, 3 clones each were inoculated; 4 ml LB+Cm (35 µg/ml)

-          Incubation ON

 

New plasmid preparation for parts 1 – 4

-          Inoculation of parts 1, 3, 4 C1 and part 2 C2 in 4 ml LB+Amp

-          Incubation ON

Re-streak clones for parts 6 and 7 on 35 µg/ml Cm plates + incubation on

Purification of part 8 (RD) products from today

PCR clean-up with Qiagen kit

 

Elution with 50 µl HPLC water (pre-warmed)

NanoDrop:

	Concentration (ng/µl)	A260nm/A280nm	A260nm/A230nm
part 8 purification	7.5	1.34	1.39

 

 

 

Either just low amounts of DNA in the sample or the purification was not very efficient. Eluded a second time

with 20µl, no DNA found.