Difference between revisions of "GFP Protocol Jugaad"

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 +
 
== '''Materials''' ==
 
== '''Materials''' ==
  
Line 85: Line 86:
 
'''Revival of Host'''
 
'''Revival of Host'''
  
1. Break open the lyophilized vial, add 0.1 ml of LB
+
'''1.''' Break open the lyophilized vial, add 0.1 ml of LB
 
broth.
 
broth.
  
2. Streak a loopful of the suspension onto LB plate &culture tubes.
+
[[File:MG_6822.JPG]]  [[File:MG_6833.JPG]]
 +
 
 +
'''2.''' Streak a loopful of the suspension onto LB plate & LB culture tubes.
 
(in duplicates).
 
(in duplicates).
  
[[File:MG_6792.JPG]]  [[File:MG_6799.JPG]]  [[File:MG_6808.JPG]]  [[File:MG_6837.JPG]]     [[File:MG_6887.JPG]]  [[File:MG_6898.JPG]]  [[File:MG_6841.JPG]]  [[File:MG_6842.JPG]][[File:MG_6899.JPG]]  [[File:MG_6901.JPG‎]]
+
[[File:MG_6792.JPG]]  [[File:MG_6799.JPG]]   
 +
 
 +
[[File:MG_6808.JPG]]  [[File:MG_6837.JPG]]
 +
 
 +
[[File:MG_6841.JPG]]  [[File:MG_6842.JPG]]   
 +
 
 +
[[File:MG_6859.JPG]][[File:MG_6843.JPG]]   
 +
 
 +
[[File:MG_6881.JPG]]
 +
 
 +
 
 +
 
 +
'''3.''' Incubate the plates at 37°C, overnight.
 +
 
 +
[[File:MG_6887.JPG]]  [[File:MG_6898.JPG]]
 +
 
 +
[[File:MG_6899.JPG]]  [[File:MG_6901.JPG‎]]
  
  
[[File:MG_6843.JPG]]  [[File:MG_6881.JPG]]
 
  
3. Incubate the plates at 37°C, overnight.
 
  
[[File:MG_6841.JPG]]  [[File:MG_6842.JPG]]  [[File:MG_6859.JPG]]
 
  
 
'''Ligation of Vector to Insert'''
 
'''Ligation of Vector to Insert'''
  
4. Thaw the ligase assay buffer, vector and insert DNA.
+
'''4.''' Thaw the ligase assay buffer, vector and insert DNA.
  
 
'''Note:''' Thaw the ligase assay buffer vial on ice,
 
'''Note:''' Thaw the ligase assay buffer vial on ice,
 
store at -20°C immediately after use.
 
store at -20°C immediately after use.
  
5. Set up ligation reaction as follows:
+
'''5.''' Set up ligation reaction as follows:
  
Water : 11 μl
+
-Water : 11 μl
  
Vector DNA : 2 μl
+
-Vector DNA : 2 μl
  
Insert DNA : 4 μl
+
-Insert DNA : 4 μl
  
Ligase assay buffer : 2 μl
+
-Ligase assay buffer : 2 μl
  
T4 DNA ligase : 1 μl
+
-T4 DNA ligase : 1 μl
  
 
Mix the contents by tapping gently and incubate at
 
Mix the contents by tapping gently and incubate at
Line 123: Line 139:
  
 
'''Note:''' Set up five ligation reactions simultaneously.
 
'''Note:''' Set up five ligation reactions simultaneously.
 +
 +
[[File:MG_6927.JPG]]  [[File:MG_6937.JPG]]
 +
 +
[[File:MG_6955.JPG]]
 +
 +
  
  
Line 131: Line 153:
 
'''Preparation of Competent Cells'''
 
'''Preparation of Competent Cells'''
  
6. Pick 10-12 moderate sized colonies from the LB plate
+
'''6.''' Incubate the culture tubes at 37°C. Grow until OD A600
and inoculate into 100 ml of LB broth (in a 1 litre
 
conical flask).
 
 
 
7. Incubate at 37°C, in a shaker. Grow until OD A600
 
 
reaches 0.3, this takes about 2-3 hours.
 
reaches 0.3, this takes about 2-3 hours.
  
8. Chill the culture flask on ice for 10-20 minutes.
+
'''7.''' Chill the culture tubes on ice for 10-20 minutes.
  
9. Transfer the culture aseptically into sterile centrifuge
+
'''8.''' Transfer the culture aseptically into sterile vials. (5 vials with 0.5ml each)
tubes and spin down at 6000 rpm for 8 minutes,
+
 +
'''9.''' Spin down at 6000 rpm for 8 minutes,
 
preferably in a refrigerated centrifuge at 4°C or spin at
 
preferably in a refrigerated centrifuge at 4°C or spin at
 
Room temperature (RT).
 
Room temperature (RT).
  
10. Discard the supernatant.
+
'''10.''' Discard the supernatant.
  
11. Resuspend the cell pellet very gently in small volume
+
'''11.''' Resuspend the cell pellet very gently in small volume
 
of ice-cold solution A (approximately 2 ml), using a
 
of ice-cold solution A (approximately 2 ml), using a
 
pre-chilled pipette. Care must be taken not to remove
 
pre-chilled pipette. Care must be taken not to remove
Line 153: Line 172:
 
33 ml of solution A, resuspend gently.
 
33 ml of solution A, resuspend gently.
  
12. Keep the centrifuge tubes on ice for 20 minutes.
+
'''12.''' Keep the vials on ice for 5 minutes.
Centrifuge at 3500 rpm for 15 minutes at 4°C or spin at
+
Centrifuge at 8000 rpm for 5 minutes at 4°C or spin at
 
RT.
 
RT.
  
13. Discard the supernatant and chill the tube on ice.
+
'''13.''' Discard the supernatant and chill the tube on ice.
 
Resuspend the pellet in 3 ml of ice-cold solution A.
 
Resuspend the pellet in 3 ml of ice-cold solution A.
  
Line 163: Line 182:
 
are very fragile at this stage.
 
are very fragile at this stage.
  
14. Aliquot 0.6 ml of the above suspension into 5 different
+
[[File:MG_7020.JPG]]  [[File:MG_7028.JPG]]
pre-chilled 1.5 ml vials, aseptically.
+
 
 +
[[File:DSC02293.JPG]]  [[File:MG_7052.JPG]]
  
15. Heat inactivate the ligated samples at 65°C for
+
[[File:MG_7075.JPG‎]]  [[File:MG_7083.JPG]]
 +
 
 +
[[File:DSC02295.JPG]] [[File:MG_7052.JPG]]
 +
 
 +
[[File:MG_7174.JPG]]
 +
 
 +
 
 +
 
 +
'''14.''' Heat inactivate the ligated samples at 65°C for
 
10 minutes. Spin at 5000 rpm for 2 minutes and keep
 
10 minutes. Spin at 5000 rpm for 2 minutes and keep
 
the vials on ice.
 
the vials on ice.
  
16. Aliquot 2 μl (10 ng) of control DNA each into 5 prechilled
+
'''15.''' Aliquot 2 μl (10 ng) of control DNA each into 5 ligation vials
1.5 ml vials.
+
 
 +
[[File:MG_7176.JPG]]    [[File:MG_7177.JPG]]
 +
 
 +
[[File:DSC02296.JPG]]  [[File:MG_7174.JPG]]
 +
 
 +
[[File:MG_7163.JPG]]    [[File:MG_7028.JPG]]
 +
 
 +
 
 +
 
  
  
 
'''Transformation:'''
 
'''Transformation:'''
  
'''Note:''' Use single aliquot of competent cells
 
(0.6 ml) for one set of transformation experiment.
 
  
17. Take a single vial of ligated sample and control DNA.
+
'''16.''' Take a 2 vials of ligated sample and control DNA and 1 vial of only control DNA( positive control).
 
To this add 200 μl each of competent cells. Tap the
 
To this add 200 μl each of competent cells. Tap the
 
vials gently and incubate on ice for 20 minutes.
 
vials gently and incubate on ice for 20 minutes.
12 13
 
  
18. Label the remaining 200 μl of competent cells as nontransformed
+
 
 +
'''17.''' Label the remaining 200 μl of competent cells as non transformed (negative control)
 
cells, place on ice till the plating step (step
 
cells, place on ice till the plating step (step
24).
+
23).
  
19. Tap all the vials gently and incubate on ice for
+
'''18.''' Tap all the vials gently and incubate on ice for
 
20 minutes.
 
20 minutes.
  
20. Heat shock the cells by placing the vial(s) in 42°C water
+
'''19.''' Heat shock the cells by placing the vial(s) in 42°C water
 
bath for 2 minutes, then return the vials to ice and chill
 
bath for 2 minutes, then return the vials to ice and chill
 
for 5 minutes.
 
for 5 minutes.
  
21. Add 0.5 ml of LB broth aseptically to the vial(s) and
+
'''20.''' Add 0.5 ml of LB broth aseptically to the vial(s) and
 
incubate at 37°C (shaker) for an hour. This is to allow
 
incubate at 37°C (shaker) for an hour. This is to allow
 
bacteria to recover and express the protein.
 
bacteria to recover and express the protein.
  
22. Pipette 200 μl each from the vials transformed with the
+
'''Note:''' While the heat shock treatment is going on, prepare the LB-Amp agar(200 μl in 200 ml of LB agar.)
 +
 
 +
[[File:MG_7064.JPG]]    [[File:MG_7133.JPG]]
 +
 
 +
[[File:MG_7142.JPG]]    [[File:MG_7143.JPG]]
 +
 
 +
[[File:MG_7174.JPG]]    [[File:MG_7175.JPG]]
 +
 
 +
[[File:MG_7177.JPG]]    [[File:MG_7174.JPG]]
 +
 
 +
[[File:MG_7185.JPG]]    [[File:MG_7191.JPG]]
 +
 
 +
[[File:MG_7190.JPG]]    [[File:MG_7186.JPG]]
 +
 
 +
[[File:DSC02301.JPG]]
 +
 
 +
 
 +
 
 +
'''21.''' liquify the already made LB-amp agar and pour about 20 to 25 ml of i in each plate.
 +
 
 +
'''Note:''' Wait for agar to solidify again.
 +
 
 +
'''22.''' Pipette 200 μl each from the vials transformed with the
 
ligated mix onto LB-Amp plates and spread thoroughly
 
ligated mix onto LB-Amp plates and spread thoroughly
 
using spreader/pipette.
 
using spreader/pipette.
  
23. Pipette 200 μl of LB onto a LB -Amp plate and spread
+
'''23.''' Pipette 200 μl of LB onto a LB -Amp plate and spread
 
20 ìl of the cells transformed with control DNA. Label
 
20 ìl of the cells transformed with control DNA. Label
this as control plate.
+
this as positive control plate.
  
24. Plate 200 μl of non-transformed cells onto another
+
'''24.''' Plate 200 μl of non-transformed cells onto another
 
LB-Amp plate to check for contamination. Label this
 
LB-Amp plate to check for contamination. Label this
as non-transformed plate.
+
as non-transformed (negative control) plate.
 +
 
 +
'''25.''' Keep one plate with only LB-Amp Agar and nothing else. Name this as double negative control.
  
25. Incubate the plates at 37°C, overnight.
+
'''26.''' Incubate the plates at 37°C, overnight.
  
  
 +
[[File:MG_7204.JPG]]    [[File:MG_7206.JPG]]
 +
 +
[[File:MG_7213.JPG]]    [[File:MG_7221.JPG]]
 +
 +
[[File:MG_7225.JPG]]    [[File:3.JPG]]
 +
 +
[[File:4.JPG]]          [[File:5.JPG]]
 +
 +
[[File:6.JPG]]          [[File:7.JPG]]
  
 
=== Day 3: ===
 
=== Day 3: ===
Line 220: Line 287:
 
'''Moment of Truth'''
 
'''Moment of Truth'''
  
26. Observe the plates under UV-light (312 nm).
+
'''27.''' Observe the plates under UV-light (312 nm).
  
 
'''Note:''' If observed add 254 nm of less intensity of glow
 
'''Note:''' If observed add 254 nm of less intensity of glow
 
could vary.
 
could vary.
  
'''Note:''' Steps 17 to 26 explains the protocol for one set
+
'''Note:''' The 3 controls:
of transformation experiment. Carry out 5 such
+
 
transformations simultaneously.
+
'''Positive control:''' (Competent cells + Control DNA.)
 +
 
 +
-If there are no colonies it means there is something wrong with the cells.
 +
 
 +
'''Negative control:''' (only Competent cells.)
 +
 
 +
-If there are no colonies it means the cells are contaminated.
 +
 
 +
'''Double Negative control:''' (Empty LB-Amp plate.)
 +
 
 +
-If there is growth then there is external contamination.
 +
 
 +
 
 +
[[File:MG_7281.JPG]]    [[File:MG_7269.JPG]]
 +
 
 +
[[File:MG_7282.JPG]]
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
== '''Precautions:''' ==
 +
 
 +
1. Work in a closed room with all the equipment in it. Don't run around for things.
 +
 
 +
2. Keep the room clean. No eatables, foot wear allowed.
 +
 
 +
3. Run UV light for about half an hour before starting experiment.
 +
 
 +
'''Note:''' do not stand close to the UV when it is on. use a uv filter to cover all sides of the box and if posible cover the incubator and walls also.
 +
 
 +
4. Clean hands with alcohol before starting anything.
 +
 
 +
5. Before using glassware autoclave them (wrap in paper and boil in cooker for 3 wistles.)
 +
 
 +
6. All work is done in the hood to avoid external contamination.
 +
 
 +
7. Sterilize everything in alcahol and flame before use
 +
 
 +
8. Keep someone beside you to give and take things from you while working in the hood. Don't keep moving your hands in and out of the hood.
 +
 
 +
9. Keep hood closed when not in use
 +
 
 +
10. When using the water bath run it till it reaches the required temp. and then use it.

Latest revision as of 18:21, 10 December 2010

Materials

The list below provides information about the materials supplied in the kit.

The products should be stored as suggested. Use the kit within 6 months of arrival.


Materials……………………... Quantity………….store at

Ampicillin………………………..100 mg………….…..4°C

Host ………………………………1 vial……..………...4°C

Solution A………………………. 40 ml…………….….4°C

Control DNA……………………...10 μl….…………..-20°C

T4 DNA Ligase………………….…5 μl………………-20°C

10X Ligase Assay Buffer ….……10 μl……….……..-20°C

Insert………………………………20 μl………..…....-20°C

Vector DNA………………………10 μl ………….….-20°C

LB Broth……………………….....15 g ……………..…RT

Agar………………………………...5 g……..………... RT

1.5 ml vials……………………….25 Nos……….….... RT


Equipment :

-Centrifuge (preferably refrigerated)( DIY handheld centrifuge ).

-UV transilluminator (312 nm).

-Spectrophotometer.


Glassware :

-Capped centrifuge tubes.

-Conical flask.

-Petri plates.

-Test tubes.


Reagent :

-Distilled water.


Other Requirements:

-Crushed ice.

-Cuvette (of 1 cm pathlength).

-Micropipette, Tips.

-Thermometer.

-Water bath. ( DIY Water Bath )

-Incubator.(DIY Incubator )

-Sterilization Hood. ( DIY Sterlisation Hood )



Procedure:

Day 1:

Revival of Host

1. Break open the lyophilized vial, add 0.1 ml of LB broth.

MG 6822.JPG MG 6833.JPG

2. Streak a loopful of the suspension onto LB plate & LB culture tubes. (in duplicates).

MG 6792.JPG MG 6799.JPG

MG 6808.JPG MG 6837.JPG

MG 6841.JPG MG 6842.JPG

MG 6859.JPGMG 6843.JPG

MG 6881.JPG


3. Incubate the plates at 37°C, overnight.

MG 6887.JPG MG 6898.JPG

MG 6899.JPG MG 6901.JPG



Ligation of Vector to Insert

4. Thaw the ligase assay buffer, vector and insert DNA.

Note: Thaw the ligase assay buffer vial on ice, store at -20°C immediately after use.

5. Set up ligation reaction as follows:

-Water : 11 μl

-Vector DNA : 2 μl

-Insert DNA : 4 μl

-Ligase assay buffer : 2 μl

-T4 DNA ligase : 1 μl

Mix the contents by tapping gently and incubate at 16°C waterbath, overnight.

Note: Set up five ligation reactions simultaneously.

MG 6927.JPG MG 6937.JPG

MG 6955.JPG



Day 2:

Preparation of Competent Cells

6. Incubate the culture tubes at 37°C. Grow until OD A600 reaches 0.3, this takes about 2-3 hours.

7. Chill the culture tubes on ice for 10-20 minutes.

8. Transfer the culture aseptically into sterile vials. (5 vials with 0.5ml each)

9. Spin down at 6000 rpm for 8 minutes, preferably in a refrigerated centrifuge at 4°C or spin at Room temperature (RT).

10. Discard the supernatant.

11. Resuspend the cell pellet very gently in small volume of ice-cold solution A (approximately 2 ml), using a pre-chilled pipette. Care must be taken not to remove the tubes from ice during resuspension. Add remaining 33 ml of solution A, resuspend gently.

12. Keep the vials on ice for 5 minutes. Centrifuge at 8000 rpm for 5 minutes at 4°C or spin at RT.

13. Discard the supernatant and chill the tube on ice. Resuspend the pellet in 3 ml of ice-cold solution A.

Note: Resuspension is to be done gently as cells are very fragile at this stage.

MG 7020.JPG MG 7028.JPG

DSC02293.JPG MG 7052.JPG

MG 7075.JPG MG 7083.JPG

DSC02295.JPG MG 7052.JPG

MG 7174.JPG


14. Heat inactivate the ligated samples at 65°C for 10 minutes. Spin at 5000 rpm for 2 minutes and keep the vials on ice.

15. Aliquot 2 μl (10 ng) of control DNA each into 5 ligation vials

MG 7176.JPG MG 7177.JPG

DSC02296.JPG MG 7174.JPG

MG 7163.JPG MG 7028.JPG



Transformation:


16. Take a 2 vials of ligated sample and control DNA and 1 vial of only control DNA( positive control). To this add 200 μl each of competent cells. Tap the vials gently and incubate on ice for 20 minutes.


17. Label the remaining 200 μl of competent cells as non transformed (negative control) cells, place on ice till the plating step (step 23).

18. Tap all the vials gently and incubate on ice for 20 minutes.

19. Heat shock the cells by placing the vial(s) in 42°C water bath for 2 minutes, then return the vials to ice and chill for 5 minutes.

20. Add 0.5 ml of LB broth aseptically to the vial(s) and incubate at 37°C (shaker) for an hour. This is to allow bacteria to recover and express the protein.

Note: While the heat shock treatment is going on, prepare the LB-Amp agar(200 μl in 200 ml of LB agar.)

MG 7064.JPG MG 7133.JPG

MG 7142.JPG MG 7143.JPG

MG 7174.JPG MG 7175.JPG

MG 7177.JPG MG 7174.JPG

MG 7185.JPG MG 7191.JPG

MG 7190.JPG MG 7186.JPG

DSC02301.JPG


21. liquify the already made LB-amp agar and pour about 20 to 25 ml of i in each plate.

Note: Wait for agar to solidify again.

22. Pipette 200 μl each from the vials transformed with the ligated mix onto LB-Amp plates and spread thoroughly using spreader/pipette.

23. Pipette 200 μl of LB onto a LB -Amp plate and spread 20 ìl of the cells transformed with control DNA. Label this as positive control plate.

24. Plate 200 μl of non-transformed cells onto another LB-Amp plate to check for contamination. Label this as non-transformed (negative control) plate.

25. Keep one plate with only LB-Amp Agar and nothing else. Name this as double negative control.

26. Incubate the plates at 37°C, overnight.


MG 7204.JPG MG 7206.JPG

MG 7213.JPG MG 7221.JPG

MG 7225.JPG 3.JPG

4.JPG 5.JPG

6.JPG 7.JPG

Day 3:

Moment of Truth

27. Observe the plates under UV-light (312 nm).

Note: If observed add 254 nm of less intensity of glow could vary.

Note: The 3 controls:

Positive control: (Competent cells + Control DNA.)

-If there are no colonies it means there is something wrong with the cells.

Negative control: (only Competent cells.)

-If there are no colonies it means the cells are contaminated.

Double Negative control: (Empty LB-Amp plate.)

-If there is growth then there is external contamination.


MG 7281.JPG MG 7269.JPG

MG 7282.JPG




Precautions:

1. Work in a closed room with all the equipment in it. Don't run around for things.

2. Keep the room clean. No eatables, foot wear allowed.

3. Run UV light for about half an hour before starting experiment.

Note: do not stand close to the UV when it is on. use a uv filter to cover all sides of the box and if posible cover the incubator and walls also.

4. Clean hands with alcohol before starting anything.

5. Before using glassware autoclave them (wrap in paper and boil in cooker for 3 wistles.)

6. All work is done in the hood to avoid external contamination.

7. Sterilize everything in alcahol and flame before use

8. Keep someone beside you to give and take things from you while working in the hood. Don't keep moving your hands in and out of the hood.

9. Keep hood closed when not in use

10. When using the water bath run it till it reaches the required temp. and then use it.