Difference between revisions of "GFP Protocol Jugaad"

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'''7.''' Chill the culture tubes on ice for 10-20 minutes.
 
'''7.''' Chill the culture tubes on ice for 10-20 minutes.
  
'''8.''' Transfer the culture aseptically into sterile vials, 5 vials with 5ml and 5 vial with 10ml
+
'''8.''' Transfer the culture aseptically into sterile vials. (5 vials with 0.5ml each)
 
   
 
   
 
'''9.''' Spin down at 6000 rpm for 8 minutes,
 
'''9.''' Spin down at 6000 rpm for 8 minutes,
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33 ml of solution A, resuspend gently.
 
33 ml of solution A, resuspend gently.
  
'''12.''' Keep the centrifuge tubes on ice for 5 minutes.
+
'''12.''' Keep the vials on ice for 5 minutes.
Centrifuge at 8000 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.
  
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'''Note:''' Resuspension is to be done gently as cells
 
'''Note:''' Resuspension is to be done gently as cells
 
are very fragile at this stage.
 
are very fragile at this stage.
 +
 +
[[File:MG_7020.JPG]]  [[File:MG_7028.JPG]]
 +
 +
.                      [[File:MG_7052.JPG]]
 +
 +
[[File:MG_7075.JPG‎]]  [[File:MG_7083.JPG]]
 +
 +
.                      [[File:MG_7052.JPG]]
 +
 +
[[File:MG_7174.JPG]]
 +
 +
  
 
'''14.''' Heat inactivate the ligated samples at 65°C for
 
'''14.''' Heat inactivate the ligated samples at 65°C for
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the vials on ice.
 
the vials on ice.
  
'''15.''' 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:MG_7174.JPG]]
 +
 
 +
[[File:MG_7028.JPG]]
 +
 
  
  
 
'''Transformation:'''
 
'''Transformation:'''
  
'''Note:''' Use single aliquot of competent cells
 
(0.6 ml) for one set of transformation experiment.
 
 
'''
 
'''
16.''' 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
 
  
'''17.''' 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).
  
 
'''18.''' Tap all the vials gently and incubate on ice for
 
'''18.''' Tap all the vials gently and incubate on ice for
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'''22.''' Pipette 200 μl of LB onto a LB -Amp plate and spread
 
'''22.''' 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.
  
 
'''23.''' Plate 200 μl of non-transformed cells onto another
 
'''23.''' 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.
  
'''24.''' Incubate the plates at 37°C, overnight.
+
'''24.''' 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.
  
  
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'''Moment of Truth'''
 
'''Moment of Truth'''
  
'''25.''' Observe the plates under UV-light (312 nm).
+
'''26.''' 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 25 explains the protocol for one set
 
of transformation experiment. Carry out 5 such
 
transformations simultaneously.
 

Revision as of 07:42, 4 September 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

. MG 7052.JPG

MG 7075.JPG MG 7083.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

. MG 7174.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.

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

22. 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.

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

24. 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.


Day 3:

Moment of Truth

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

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