Team:METUHS-Ankara/project.html

Transformation again!

Selin Hoca Transformation

Pet28a

Pet28a+gene

RFP+ DT

We cannot insert the plasmid!! Something’s wrong with the competent cells!!

Comp 1 -

Comp 2  -     RFP + DT    V   Pet28a

Comp 3  -    Amp, Kan        Kan

The second transformation.

Competent cell, why didn’t they work?

1) Lack of CaCl2

2) CaCl2 wasn’t cold enough

3) Non homogeneous cells (The closest)

4) OD was not enough

Transformation, why didn’t it work?

1) Heat shock

2) Cell died in centrifuge

3) Cell died whilst spreading

• Heat

• Mechanic force

Plasmid Isolation

.Gel Electrophoresis 10 µL                                           .Stock 15 µL

 Gel Extraction

A1         K1          RFP

Needed the plasmids!

Plasmid Isolation - 2 µL cell pellet, lyse down the cells take plamids!

                                                                                                          16.05.2014

Transformation

              pCooM       with changes of 50 µL cell

              pCooF          + 10 µL  plasmid

Digestion

NEB Buffer   5 µL

BSA              0.5 µL

Enzyme 1     0.5 µL

Enzyme 2     0.5 µL µL

Plasmid       1000 ng/µL

To complete to 50 µL, add ddH2O/dH2O

Keep it in water bath for 2.5 hours at 37ºC

LIGATION

10 x T4 ligase buffer                      2 µL

6:1 M ratio to invert to vector

Add dH2O

T4 ligase                                         1 µL

Incubate at room temtperature  1 hours and 15 min. at 65ºC for enzyme activation

Alternative: +4ºC over night

Calculatory Insert Amount

Insert mass in ng = 6 x  insert length in 6 µL / Vector length 6 g...

  ...x vector mass in mg

-Gel

-Gel Extraction

Vector - 2500 bp

Insert  - 500 bp

1300 - to prepare the agarose gel 1.95 g of agarose (nearly 2%)

-Gel Results

-PCR

-Gel

Why didn’t it result correct?

There was no DNA

Problem with gel

Part we don’t want in the gel

Lack of the amount

TAE?

Agarose

6x loading dye

Plasmid  -  Digestion, Ligation

EtBr

DNA degredation

Conditions

Initial denaturation   -   94ºC     -30 sec

Cycles                          94ºC      10-30 sec

(30-35)                        45-65ºC (Tm= 58 ºC) 15-60 sec                     2000+1800 bp

(32-33)                        65ºC   50 sec/kb (⁓2 min)

Final extension          65ºC  10 min

Hold                            +4ºC

8F 9R    /   10F 11R - primers used!

Nutrient agar plate inoculation

RFP                     GFP

IPTG +

IPTG -

Nutrient agar plate inoculation

IPTG + and –

Inoculation on broth media

IPTG x2

MP x4

​​• IPTG induction in Broth Media (1)• Miniprep pCooM, pCooF (1)                 Holin- Antiholin• CODH from BCG PCR (2)• Kill Switch Transformation (3)                  MazF- AntimazF• Digestion & Ligation (?)           BCG -primer(Rhodospirillum rubrum  primer)           

Transformation of kill switch (with changes of LB - 150 µL at last step)

Gel Extraction

Genomic DNA Isolation

Chl & Tet Inoculation

    Empty: MazF, AntimazF (streak plates) (4 inoculations)

Genomic DNA Isolation

• Suspend colonies in 120 µL TEN Buffer

                                                        40 µM Tris, 1 µM EDTA, 150 µM NaCl

• Incubate at 100ºC for 10 min

• Centrifuge at 13.000 rpm

• Take supernatant store at +4ºC

Gel Extraction

• Slice the gel

• Add 1:1 volume binding buffer

• Incubate 50-60ºC for 10 min.

• Transfer up to 800 µL of the solubilized gel solution to the purification column

• Cent. 1 min, discard flow-through

• 100 µL binding buffer cent. 1 min, discard flow-through

• 700 µL wash buffer, cent. 2 min discard flow-through

• Column to eppendorf 50 µL Elution buffer dH2O, cent. 1 min.

• Discard column store it at -20ºC

1) PCR

2) Gel Electrophoresis - Genomic DNA + Gel Extraction

-PCR

L    Uncut 2    2.2   2.1   Uncut 4   4   BA   BK    L   2T   4T    4L

                                 = Gel Electrophoresis

Gel Extraction

Nutrient Agar plate inoculation

LB at the last step of transformation is 150 µL

Ligation:

Same with 13.06.2014 but overnight +4ºC incubation  prepared at 19.45

17.06.2014 - at 11:45, will be transformed.

17.06.14 - Transformation

                    100 - 10

                    50   - 5

Transformation - heat shock 75’’

                              100   LB at the last step

Selin inoculation - amount of LB was more than needed.

0.5 eppendorf - it was opened before centrifuge (contamination)

4F   9R - primers                                             1- 2 GE               3- 2.2

PCR - 2. Sample                                              2- 2.1

LIGATION

T4 ligase                                1 µL

T4 ligase buffer                    1 µL

Vector                                   4     6     2     5     3

Insert                                    4     2     6     3     5

                                             1:1   3:1  1:3  5:3   3:5

16ºC     30’

RT        30’

RT        30’

+4ºC    Overnight

Transformation:

Öykü: 1st: (3) 2 GE 2h A (3:1)  -  2nd: 2 GE A 301

Can (Öykü): 1st: (4) 2 GE A 2h (1:3) - 2nd: 2 GE A 301

Almira: 1st: (2) 2GE A 2h 1:1 (3:5) - 2nd: 2 GE A 301 – (5) 2GE A 301

Martin: 1st: (1) 2GE A 2h 5:3 (1:1) - 2nd: 2 GE A 301

Transformation - +4 ONC

PCR - 2 - 2’

PCR Purification

Digestion

Gel

Gel Extraction

Ligation 16ºC   1h

               RT     2h

               +4     ONC

Gel

Gel Extraction

Transformation