Contents 1 2014.6.12, 10:00 1.1 bacteria transformation 2 2014.6.13 2.1 LB media composition 3 2014.6.18 4 2014.6.19 5 2014.6.21 5.1 plasmid 5.2 protocol 6 2014.6.21 7 2014.6.22 7.1 pladmid extract 7.2 Electrophoresis &extract plasmid 8 2014.7.16, 4:00 8.1 group 8.2 results 8.3 repeat experiment 9 2014.7.18 10 2014.7.19 11 2014.7.20 12 2014.7.21 12.1 2:45pm 12.2 4:00pm 13 2014.7.22,8:24 14 2014.7.24,11:00am 15 2014.7.25 15.1 8:00am 15.2 15:30pm 16 2014.7.27

experiment training& plasmid building

To build the plasmid

2014.6.12, 10:00

bacteria transformation

‘’protocol’’

1. add 2ul plasmid into the active bacteria(100 ul)
2. incubate on ice for 20min
3. 42°C water bath for 20 min
4. ice, 2 min
5. add 500ul LB
6. 37°C shake, 14 min , 100 rpm
7. 3000 rpm, 2 min
8. incubate
   • plate streaking
   • plate pouring(100ul)
   • liquid incubate(10ul)
9. incubate in 37°C incubator overnight
10. liquid medium in shaker 36.4°C, 200rpm

2014.6.13

LB media composition

‘’protocol’’

• 1L LB medium
  • 950ml ddH2O
  • 10g tryptone
  • 5g yeast extract
  • 10g sodium chloride
  • 15g agar
  • PH 7.0

2014.6.18

Sterilization 121°C 20min

2014.6.19

results

2014.6.21

select the success of transformed plasmid

plasmid

protocol

1. take out the competent cell from -80.2°C, and put with plamid in ice until they melt.(about 5min)
2. put the plasmid into the cell, and put it on the ice for about 30min
3. 42°C water bath for 90sec
4. ice deal for 2min
5. add LB in the this system(500ul)
6. shake in the shaker for 30min
7. spread plate

2014.6.21

Select the colony of mLDHA-PGEM7 and PEFM3C into the liquid medium
shake for 16hrs

2014.6.22

pladmid extract

Use the tiangen mini extract plasmid kit to extract plasmids, and got 16704ng/ul

Electrophoresis &extract plasmid

protocol

1. component of gel
   1. 1% agarose(0.41g)
   2. TAB 41ml
   3. golden gel 0.5ul
   4. add on the ice for 30min(without any bubble)
2. use kit(Tiangen) to make buffer
   • 5XM3C+5XmcDHA, add bacteria into the tube, then add P2 to mix up
3. centrifuge 2minX2
4. P1(150ul), P2(150ul), P3(150ul), store at 4°C, centrifuge 5min
5. silica gel column, 600ul supernatant, centrifuge 30sec
6. pour the percolate, add PWT 300ul, centrifuge for 1min, pour out percolate, add nothing but centrifuge for 2min to remove the alcohol.
7. add 50ul TB buffer, centrifuge for 1min, keep the percolate, put on the ice.
8. make the 50X TAE buffer, and add TAE, drown the gel.
9. add marker 3ul,
10. DNA loading buffer(1 drop)+plasmid extract(20ul)
11. test the concentration by the nanodrop

2014.7.16, 4:00

group

Bacteria 50ul per group, and plasmid:bacteria is 3%

results

Only the antibiotics-free(0) group grow up bacteria

repeat experiment

Shake bacteria about 20hrs
incubate in incubator about 16hrs

All of above do not have fluorescence

2014.7.18

gradient antibiotics&single colonies Make 100ml liquid LB medium

2014.7.19

Extract the plasmid of K608008 and do the restriction by restriction enzyme

2014.7.20

1. make 0 and A+ LB liquid medium
2. plasmid data(following table)
3. transformation of biobricks
   1. add 20ul ddH2O into the hole in the plate
   2. get 5ul plasmid
   3. use the transformation protocol
   4. spread plate 50ul

2014.7.21

2:45pm

C+ J23115 +(only one grew up)

4:00pm

2014.7.22,8:24

Note: the concentration of I20260 is less than 5ul.

2014.7.24,11:00am

‘’explore the situation suitable for transformation of biobricks’’ K608008, C+
The heat-streak time

2014.7.25

8:00am

Blank

15:30pm

All have the single colony but the same

2014.7.27

The plasmid building