Team:UCL/Labbook

June

Week 1-3

No lab work

Week 4

26th June - We are given safety training in the Advanced Centre for Biochemical Engineering in all relevant laboratories, as well as general procedures in case of emergency.

July

Week 5-6

No lab work

Week 7

Bacterial Lab

15th July - The team is introduced to the laboratories which will be used during the summer for both bacterial and mammalian experiments.

16th July - 5X M9 salts [link to protocol], minimal agar [link to protocol], 1.4% molten agar solution [link to protocol] and 0.1M CaCl2/15% glycerol [link to protocol] were prepared for the generation of competent cells. Minimal agar plates were poured and streaked [link to streaking protocol] with W3110 Escherichia coli cells and left overnight to incubate at 37C.

17th July - Very little colony growth was observed from W3110 E.coli streaked plates. Plates were therefore left to incubate for a further 17 hours.

18th July - Sufficient colony growth allowed for the selection of a single colony from each plate. This was then inoculated in 5ul LB media [link to LB recipe] + 100ul 1M MgSO4 and left to incu-shake overnight at 37C.

19th July - Cultures re-suspended in new LB media and 100µl aliquots placed into individual eppendorf tubes for placement at -80C.

Mammalian Lab

17th July - Mammalian cell culture and maintenance [link to mammalian protocol] training by Mrs. Ludmilla Ruban. Passaged primary MEF (mouse embryonic fibroblast) cells. Passage 3.

18th July - MEF passage 4

19th July - MEF passage 5

Week 8

Bacterial Lab

22nd July -Transformation [link to transformation protocol] of our competent cells with plasmid YB3110 was carried out.

23rd July - No colony growth was observed on Ampicillin plates [link to ampicillin plate protocol] indicating no plasmid uptake. Transformation was repeated with YB3110.

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	0
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	0

24th July - No colony growth from main experiment indicating no plasmid uptake. Transformation was repeated.

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	0
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	0

25th July - No colony growth and hence no plasmid uptake. Transformation was repeated.

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	0
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	0

26th July - Once again our cells were unsuccessful in taking up the YB3110 plasmid. We concluded that our batch of competent cells were not competent and so these will not be used in any further experiments. A new batch of competent cells are to be generated.

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	0
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	0

Mammalian Lab

22nd July - MEF passage 6. Training ends.

Week 9

Bacterial Lab

29th July - A glycerol stock of pSecTag2A from 2009 was restored. This was streaked onto 3 plates (2x LB Amp and 1x No drug), additionally four Falcons with 2ul LB were inoculated with the glycerol stock (2x LB No drug, 2X LB Amp). These were left to incubate overnight.

30th July - pSecTag2A Amp and No drug cultures were centrifuged (5 minutes at 4000rpm) and the pellet frozen to be used later in a miniprep. pSecTag2A plates displayed good colony growth. Colonies were picked from non-competent W3110 streaked plates and inoculated in LB ->incu-shake 37C o/n.

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	100+
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	0

31st July - A new stock of competent cells was generated and stored, these were tested for competence via transformation using pSecTag2A and streaking onto amp plates -> incubate 37C o/n.

Ampicillin [link to Ampicillin protocol] was produced and stored. Following miniprep of pSecTag2A [link to miniprep anachem protocol] a gel [link to gel protocol] was prepared for analytical digest with HindIII [link to HindIII conditions] [insert gel image HindIII analytical digest of pSecTag2A]. 50X TAE diluted to 1X [link to dilution protocol].

Item	Volume (ul)
DNA pSecTag2A	5
HindIII	1
Buffer	1
BSA	0.5
dH20	2.5
Total	10

Results displayed uncut bands at expected lengths. However HindIII cut pSecTag2A wells displayed too many bands, indicating possible contamination or uncut DNA.

August

1st August - Results from newly generated competent cells transformed with pSecTag2A:

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	100+
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	10

This indicated that the cells are more competent than the last batch, although growth on the negative control was a cause for concern. Therefore the transformation procedure from yesterday was repeated to see whether the ampicillin was working -> plates left for incubation 37C o/n.

Transformation of competent cells with pSecTag2A. 100µl was then spread onto 6 plates (4x Amp, 2x No drug). Left to incubate o/n 37C.

2nd August - Plates displayed significant colony growth on Amp plates indicating successful transformation. Negative control however displayed slight colony growth - ampicillin not working effectively, new ampicillin was therefore prepared. End of week inventory was recorded and stocks were topped up.

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	100+
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	15

Mammalian Lab

29th July - Thawed [internal link to mammalian protocol] and revived HeLa cells, grown in two T25 flasks in DMEM + 10% FBS + 2 mM L-Glu

30th July - HeLa cells are not looking very happy, with many floating cells. Cells are left to grow over the weekend

Week 10

Bacterial Lab

5th August - The new batch of ampicillin was tested via transformation of competent cells with pSecTag2A. Plates were spread and incubated 37C o/n.

6th August - Results from yesterday’s transformation:

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	100+
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	15

This indicates that there is still an issue with the ampicillin, possibly a problem with the stock powder used. A final test with both old and new ampicillin was carried out and compared without plasmid insertion.

7th August -

Vial	Ampicillin Plate	Plasmid Insertion	Colony Count
1 (Main)	Yes	Yes	100+
2 (Positive Control)	No	Yes	100+
3 (Negative Control)	Yes	No	15

Results indicated that the ampicillin source may not have been fully functional, therefore a new source of amp powder was located and amp was remade.

8th August - Preparation of 4x Amp plates and 4x no drug plates for storage in the fridge.

9th August - Meeting with Darren Nesbeth, requirements for upcoming weeks: create glycerol stocks of both pSecTag2A and pSB1C3, purify and extract pure DNA for pSecTag2A and pSB1C3.

Mammalian Lab

6th August - Checked HeLa cells. Cells are growing slow, left to grow for a few more days

8th August - HeLa cells are about 30% confluent. Changed media.