Team:Hong Kong CUHK/june

Jun 20 Mary, Jennifer

* Promoters and plasmids : 11A, 18C, 11A_psB 1AH3, 18C_BBa_J61002

Add 10 H2O to 1

*Transformation

ice bath 30min-->

42oC for 90 seconds--(+LB)---> 

incubate while shaking 1hour

*LB agar: 10g LB, Agarose 9.5g, Overall volume:500ml

Jun 24 Eddie , Ar Sze

*25X TAE buffer made(1 Litre)

*streak DH5X and BL21 plates for competent cells making (in incubator)

*CCMB80 buffer 0.5L

*Stephen demed picking colonies, will put clips on FB

Highlights for 25/6/2013

*pick colonies according to Day 0 on whiteboard

*Transformation of T7+RBS according to whiteboard

*SP* Autoclave Eppendoff and buffer and then put in the fridge

Eppendoff in -20 oC buffer ice cold {pre-cool}

Do not erase whiteboard

Shake Medium 12-16 hours--------→too long would make colonies too large

Incubator 16-20 hours -------------------------------------↑

Jun 25 Eric, George, Isaac, Maurice, Jennifer, Kelton, Natalie, Gill, Cheng, Eddie, Roy

* pick colonies ( competent cells making DH5 , BL21)

* transformation (T7+RBS)

* miniprep demonstration

* agar plate preparation x 6

Eddie and Melisa remake 200ml CCMB80 buffer in 4 falcon

Melisa brought 18 pipette up to lab.

Hightlight for tomorrow: complete making of competent cells, finish transformation of T7+RBS, may try miniprep of that.

Jun 26 Eric, Jackie, Maurice, George, Eddie, Chris

1. Finished with transformation of pSB1K3-RFP plasmid. Total 120ml obtained. Ran gel and confirmed correct plasmid obtained. Found that at least 20 ml TAE is needed.

2. Picked colonies from T7-RBS plate, incubation for miniprep later.

3. Finish making competent cells, but due to time constrain they were not pipetted into Eppendoff. The cells were stored in Falcon in -80oC.

4. Big Sunny brought a pipette, a gun for large volume pipette from BME lab. Used TAE should be stored in 'used TAE' bottle, and can be reused until it looks bad.

5. We made a liter of 1x TAE, 250ml used and recycled in 'Used Bottle'.

Hightlight for tomorrow:

1. Get competent cells into Eppendoff.

2. Transformation efficiency determination.

3. Minipre T7-RBS.

4. Run gel to confirm correct plasmid obtained. ( T7+RBS is small, so band should appear at very end of the gel, use E and P site for largest fragment, of about 50 bp.)

Jun 27 Maurice, Thomas, Natalie, Jackie, Eddie, Eric, Isaac, Kelton, Roy, George, Jack

1. Competent cells got into tubes. Kelton found that DH5 cells are very low in concentration. Need very long recovery in these cells.

2. T7-RBS culture was a failure, Transformation redone, incubating.

Jun 28 Maurice, Eddie, Natalie, Melisa, Roy, Eric

1. Picked colonies from DH5 and BL21 dish transformed with T7+RBS in pSB1C3. Shaking at 37oC in 250 rpm. Transformation efficiency of DH5 and BL21 is taken note. BL21 did very good, DH5 is not very good. So we can use BL21 for transformation if large number of colonies are wanted.

Jun 29

1. Restriction digestion of plasmid obtained.

2. Run gel, results are not visible for both BL21 and DH5 .

3. Miniprep for BL21 was performed again.

Miniprep of T7-RBS plasmid (psK1C3)

>Gel photo 1 (no result)