Difference between revisions of "Team:Groningen/Labjournal"

PCR

Experiment:

27/09 The key sequence was amplified see PCR protocolfrom the pDR111+key plasmid with the primers key only + prefix and key only + suffix (primer sequences can be found here). The correct size 187 bp of the product was checked by gel electrophoresis. The PCR product was stored at 4°C.

PCR mixture:

50 μl PCR assay was performed according to the following protocol: see PCR protocol

PCR set-up:

DNA Electrophoresis:

For detailed information on how to prepare and run agarose gel see following protocol.

Conclusion:

The key sequence with prefix and suffix was successfully amplified from the gBlock.

Procedure after gel validation:

PCR product was subsequently cleaned with PCR Purification Kit – Jena Bioscience see protocol

Restriction digestion

Experiment:

04/10 The key sequence PCR product was digested with EcoRI and PstI as well as the BBa_K823023 plasmid. The digestion of the BBa_K823023 backbone was checked with gel electrophoresis. The digestion of the key sequence PCR product was immediately cleaned with PCR Purification Kit – Jena Bioscience see protocol

RD mixture:

20 μl RD assay was performed according to the following protocol: RD protocol

DNA Electrophoresis:

For detailed information on how to prepare and run agarose gel see following protocol.

Conclusion:

The digestion of BBa_K823023 showed the correct bands on the gel for the 6000 bp backbone and the 1000 bp RFP insert. Therefore the cloning procedure could proceed.

Procedure after gel validation:

Digested sample of the backbone ~6000 bp were cut out from the gel and DNA was extracted by Gel extraction kit (Nucleospin) see protocol.

Ligation

Experiment:

04/10 The EcoRI and PstI cut BBa_K823023 integration backbone was ligated with the EcoRI, PstI cut key sequence.

Ligation mixture:

20 μl ligation assay was performed according to the following protocol: see protocol.

Transformation

Experiment:

04/10 The ligation mix was heat shock transformed to competent Top10 E. coli cells following the protocol. Cells were plated on 100 μg/ml ampicillin LB agar to select for the correct construct. The next day colonies were selected to perform colony PCR in order to find the correct construct.

PCR mixture:

25 μl PCR assay was performed according to the following protocol: see PCR protocol

PCR set-up:

DNA electrophoresis:

For detailed information on how to prepare and run agarose gel see following protocol.

Conclusion:

Plasmid

Experiment:

05/10 Grown cultures of E. coli Top10 with the key sequence in BBa_K823023 were used for making the glycerol stocks (see Mini prep protocol). Firstly, concentration of the plasmids obtained was measured on Nanodrop. Secondly, plasmids were sent for sequencing and then stored at -20°C.

PCR

Experiment:

25/07 Message sequence was amplified from gBlock called message sequence ordered from IDT (Integrated DNA technologies, link to sponsors?).Primers used for the amplification were F-message sequence and R-message sequence (primer sequences can be found in Primer list link).

PCR mixture:

50 μl PCR assay was performed according to the following protocol.

PCR set-up:

DNA Electrophoresis:

For detailed information on how to prepare and run agarose gel see following protocol.

Conclusion:

PCR of the message sequence from the IDT gBlock was successful. It was verified by DNA electrophoresis (see above). Correct size of a band could be seen and that was 572 bp.

Procedure after gel validation:

PCR product was subsequently cleaned with PCR Purification Kit – Jena Bioscience (see protocol).

Restriction digestion

Experiment:

26/07 On this day restriction digestion of PCR product of the message sequence (see above) and pDR111 integration plasmid was done. Message sequence as an insert was cut with SalI and HindIII restriction enzymes.Integration plasmid pDR111 as a vector was cut with exactly same restriction enzymes.

RD mixture:

20 μl RD assay was performed according to the following protocol.

DNA Electrophoresis:

For detailed information on how to prepare and run agarose gel see following protocol.

Conclusion:

RD of the PCR product of message sequence and integration plasmid pDR111 was successful. It was verified by DNA electrophoresis (see above). Correct size of bands could be seen and that was 7834 bp for pDR111 and 572 bp for message sequence.

Procedure after gel validation:

Digested samples were cut out from the gel and DNA was extracted by Gel extraction kit (Nucleospin) (see protocol).

Ligation

Experiment:

26/07 Restriction digestion (see above) was followed by overnight ligation.Vector (integration plasmid pDR111) and insert (message sequence) was ligated in 1:5 molar ratio.

Ligation mixture:

20 μl ligation assay was performed according to the following protocol.

Transformation

Experiment:

27/07 This day transformation into E. coli Top10 and MC1061 was done. Correct clones were selected on 100 μg/ml ampicillin plates. Transformation protocol used can be found here.

28/07 Next day 12 colonies were obtained on the plates of E. coli MC1061 strain (see Figure 5). To verify correct transformants colony PCR was performed (see Colony PCR protocol). Primers F-message sequence and R-message sequence were used for this colony PCR. Sequences of these primers can be found here (link to primer list).

PCR mixture:

25 μl PCR assay was performed according to the following protocol.

PCR set-up:

DNA electrophoresis:

For detailed information on how to prepare and run agarose gel see following protocol.

Conclusion:

Transformation of pDR111+message into E. coli MC1061 appeared to be successful. Transformation efficiency was low, only 12 colonies were obtained, but 4 colonies seemed to be correct clones. And those were colonies 1, 8, 10 and 11 (see Figure 5). Correct sizes of the bands were obtained: 572 bp. Those colonies were grown overnight (see Cell culture protocol) from pre-glycerol stocks made for colony PCR (see Colony PCR protocol).

Plasmid

Experiment:

29/07 Grown cultures of E. coli MC1061 with pDR111+message were taken out from the incubator after overnight incubation. Glycerol stocks were made (see protocol for glycerol stock) and plasmid isolation was performed (see Mini prep protocol). Firstly, concentration of the plasmids was measured on Nanodrop. Secondly, plasmids were sent for sequencing and then stored at -20°C.

Sequencing:

Plasmids pDR111+message from colonies 1, 8, 10 and 11 were sent for sequencing (see Sequencing protocol) with the sequencing primer Gb_insert_F2 (primer sequence can be found here primer list link).

Conclusion:

Sequencing results showed that cloning of message sequence into integration plasmid pDR111 was successful. However just sequencing result 249 (Figure 9) has 100 % homology when compared to the reference (message sequence). Others have some bases missing or bases are not matching, it could be due to faulty sequencing. See sequencing results below.

Experiments

Experiments:

Integration into B. subtilis 168 trp+ was done to integrate the key sequence into the genome of B. subtilis.

You can find the actual integration experiment here.

(Experiment title)

Experiment:

(text)

PCR mixture:

(text) (link to protocol)

PCR set-up:

DNA Electrophoresis:

(text or something)

Conclusion:

(something or text)

Procedure after gel validation:

(washed hands)