User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/05/23

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{|{{table}} width="800"
 
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">PcTF Subcloning in E. coli</span>
 
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|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
 
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<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
 
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=5/23/2012=
=5/23/2012=
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#Prepare 500mL 1X NuPAGE SDS running buffer
#Prepare 500mL 1X NuPAGE SDS running buffer
#*25mL 20X MES Running Buffer (for very small proteins e.g. 13kD) or 20X MOPS Running Buffer (for medium-sized proteins e.g. 50-60kD).
#*25mL 20X MES Running Buffer (for very small proteins e.g. 13kD) or 20X MOPS Running Buffer (for medium-sized proteins e.g. 50-60kD).
-
#*450mL deionized water
+
#*475mL deionized water
#Mix well.
#Mix well.
#Set aside 100mL buffer for use in Upper (Inner) buffer chamber).
#Set aside 100mL buffer for use in Upper (Inner) buffer chamber).
-
#*Add 400&mu;L NuPAGE Antioxidant immediately before running the gel.
+
#*Add 500μL NuPAGE Antioxidant immediately before running the gel.
#*Mix well.
#*Mix well.
===Sample preparation===
===Sample preparation===
#Wells can accommodate 25&mu;L loading volume.   
#Wells can accommodate 25&mu;L loading volume.   
-
#*''Likely they can accommodate more ... maybe 40&mu;L.''
 
#Let sample buffer warm to room temperature.
#Let sample buffer warm to room temperature.
#Each sample should contain
#Each sample should contain
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Note that prestained molecular weight marker doesn't need any preparation.
Note that prestained molecular weight marker doesn't need any preparation.
 +
 +
After the heating process keep the samples on the bench to cool down to the room temperature.
===Running the gel===
===Running the gel===
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#Fill the upper buffer chamber with a small amount of upper buffer chamber running buffer (with antioxidant) to check tightness of seal.
#Fill the upper buffer chamber with a small amount of upper buffer chamber running buffer (with antioxidant) to check tightness of seal.
#*If there is a leak, discard buffer, reseal chamber and try again.
#*If there is a leak, discard buffer, reseal chamber and try again.
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#Fill upper buffer chamber.  Buffer level should exceed level of the wells.  Requires about 200mL
+
#Fill upper buffer chamber.  Buffer level should exceed level of the wells.  Requires about 400mL
#Load samples.
#Load samples.
#Load protein molecular weight marker (20 &mu;L per lane but 10&mu;L also seems to work).
#Load protein molecular weight marker (20 &mu;L per lane but 10&mu;L also seems to work).
-
#Fill lower buffer chamber at the gap near locking mechanism with 600mL NuPAGE SDS running buffer.
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#Fill lower buffer chamber at the gap near locking mechanism until the upper part of the removed white tape with 100mL NuPAGE SDS running buffer.
-
#Run at 200V for 30 minutes.
+
#Run at 120V until the samples running down to the nearest bottom edge of the gel.
 +
 
 +
Categorization:
 +
 
 +
{| class="wikitable" width=400px
 +
| Well # || Sample
 +
|-
 +
| 1 || Protein Ladder
 +
|-
 +
| 2 || NEB-10B-BD113-AB
 +
|-
 +
| 3 || NEB-10B-BD113-AA
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|-
 +
| 4 || NEB-10B-BD112-AA
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|-
 +
| 5 || NEB-10B-BD112-AB
 +
|-
 +
| 6 || BL21-BD112-AA
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|-
 +
| 7 || BL21-BD112-AB
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|-
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| 8 || BL21-BD113-AA
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|-
 +
| 9 || BL21-BD113-AB
 +
|-
 +
| 10 || DH5α(Control)
 +
|-
 +
| 11 || Empty
 +
|-
 +
| 12 || Empty*
 +
|-
 +
|
 +
|}
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* Do not leave the wells empty, load empty wells with samples of NuPAGE LDS Buffer and Protein extraction reagent.
 +
 
===Staining the gel===
===Staining the gel===
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#Cut to separate gel from bottom lip.
#Cut to separate gel from bottom lip.
#Flip over and transfer gel to staining tray that has been prefilled with 100mL deionized water (see below).
#Flip over and transfer gel to staining tray that has been prefilled with 100mL deionized water (see below).
-
#*''Use lid of a 1000&mu;L pipette tip box.''
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#Add 100mL deionized water in staining tray.
-
 
+
#Shake staining tray for 5 min on orbital shaker at room temperature.
-
You have three options for staining at this point.
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#Discard water.
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#Repeat steps 1-3 two more times.
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#[[Knight:NuPAGE electrophoresis/Fast staining]] (fast but less sensitive protocol)
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#Add 20mL SimplyBlue SafeStain (enough to just cover the gel).
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#[[Knight:NuPAGE electrophoresis/Slow staining]] (slow but more sensitive protocol)
+
#Shake staining tray for 1 hr on orbital shaker at room temperature.
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#[[Knight:NuPAGE electrophoresis/Hybrid staining]] (allows you to see results immediately but take gel picture when the gel is stained more thoroughly)
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#*''Overnight is better for improved sensitivity.''
-
 
+
#Repeat steps 9, 10 and 11.
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See [[Knight:NuPAGE electrophoresis/Gel drying]] for instructions on how to dry and preserve the gel.
+
#Shake staining tray for 1 hr or more on orbital shaker at room temperature.
-
 
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#*''If the gel has been stained overnight, let it destain for a few hours.''
===Marker Sizes===
===Marker Sizes===

Current revision

Contents

5/23/2012

Materials

Procedure

Running buffer preparation

Do this step first.

  1. Prepare 500mL 1X NuPAGE SDS running buffer
    • 25mL 20X MES Running Buffer (for very small proteins e.g. 13kD) or 20X MOPS Running Buffer (for medium-sized proteins e.g. 50-60kD).
    • 475mL deionized water
  2. Mix well.
  3. Set aside 100mL buffer for use in Upper (Inner) buffer chamber).
    • Add 500μL NuPAGE Antioxidant immediately before running the gel.
    • Mix well.

Sample preparation

  1. Wells can accommodate 25μL loading volume.
  2. Let sample buffer warm to room temperature.
  3. Each sample should contain
    • 13 μL protein sample (max 0.5μg per band)
    • 5 μL NuPAGE LDS Sample Buffer
    • 2 μL NuPAGE Reducing Agent
  4. Heat samples at 70°C for 10 mins.

Set up the gel apparatus during the 10 mins sample heating step.

Note that prestained molecular weight marker doesn't need any preparation.

After the heating process keep the samples on the bench to cool down to the room temperature.

Running the gel

  1. Wear gloves.
  2. Remove the NuPAGE gel from the pouch.
  3. Rinse the gel cassette with deionized water.
  4. Peel the tape from the bottom of the cassette.
  5. Gently pull the comb from the cassette in one smooth motion.
  6. Rinse the sample wells with 1X NuPAGE SDS running buffer.
    • Use a pipetman and pipet to squirt in running buffer.
  7. Invert and shake to remove buffer.
  8. Repeat rinse two more times.
  9. Orient the two gels in the Mini-Cell such that the notched "well" side of the cassette faces inward towards the buffer core.
  10. Seat the gels on the bottom of the Mini-Cell and lock into place with the gel tension wedge.
    • Use the plastic buffer dam if you are only running one gel.
  11. Fill the upper buffer chamber with a small amount of upper buffer chamber running buffer (with antioxidant) to check tightness of seal.
    • If there is a leak, discard buffer, reseal chamber and try again.
  12. Fill upper buffer chamber. Buffer level should exceed level of the wells. Requires about 400mL
  13. Load samples.
  14. Load protein molecular weight marker (20 μL per lane but 10μL also seems to work).
  15. Fill lower buffer chamber at the gap near locking mechanism until the upper part of the removed white tape with 100mL NuPAGE SDS running buffer.
  16. Run at 120V until the samples running down to the nearest bottom edge of the gel.

Categorization:

Well # Sample
1 Protein Ladder
2 NEB-10B-BD113-AB
3 NEB-10B-BD113-AA
4 NEB-10B-BD112-AA
5 NEB-10B-BD112-AB
6 BL21-BD112-AA
7 BL21-BD112-AB
8 BL21-BD113-AA
9 BL21-BD113-AB
10 DH5α(Control)
11 Empty
12 Empty*
  • Do not leave the wells empty, load empty wells with samples of NuPAGE LDS Buffer and Protein extraction reagent.


Staining the gel

  1. Shut off the power.
  2. Disconnect electrodes.
  3. Remove gels.
  4. Insert a knife in between the two plates and pry the plates apart.
    • You should hear a cracking noise as you break the bond between the two plates.
  5. Gently separate the two plates attempting to leave the gel on the bottom slotted plate.
  6. Cut to separate gel from bottom lip.
  7. Flip over and transfer gel to staining tray that has been prefilled with 100mL deionized water (see below).
  8. Add 100mL deionized water in staining tray.
  9. Shake staining tray for 5 min on orbital shaker at room temperature.
  10. Discard water.
  11. Repeat steps 1-3 two more times.
  12. Add 20mL SimplyBlue SafeStain (enough to just cover the gel).
  13. Shake staining tray for 1 hr on orbital shaker at room temperature.
    • Overnight is better for improved sensitivity.
  14. Repeat steps 9, 10 and 11.
  15. Shake staining tray for 1 hr or more on orbital shaker at room temperature.
    • If the gel has been stained overnight, let it destain for a few hours.

Marker Sizes

  • The SeeBlue Plus 2 marker (blue unless another color is indicated); 10 μL samples have around 1-2 μg of protein per band
    • Myosin 188 KD (MES buffer) 191 KD (MOPS buffer)
    • Phosphorylase B 98 KD (MES buffer) 97 KD (MOPS buffer) (orange)
    • BSA 62 KD (MES buffer) 64 KD (MOPS buffer)
    • Glutamic dehydrogenase 49 KD (MES buffer) 51 KD (MOPS buffer)
    • Alcohol dehydrogenase 38 KD (MES buffer) 39 KD (MOPS buffer)
    • Carbonic anhydrase 28 KD
    • Myoglobin 17 KD (MES buffer) 19 KD (MOPS buffer) (purple)
    • Lysozyme 14 KD
    • Aprotinin 9 KD
    • Insulin B chain 3 KD

Notes

  • To date, the best staining tray I've found is the lid of a 1000μL pipette tip box. Then use a piece of mesh that just fits inside the lide to either keep the gel in place while changing solutions or to move the gel to and from the light box. This method requires smaller volumes of stain than the staining tray from Invitrogen.
  • There are several ways to screw up this protocol, I list them here in the hopes that it may help people avoid these trivial mistakes.
    1. Failing to remove the seal at the bottom of the precast gel
    2. The inner and outer chambers of running buffer are not sealed from one another.
    3. Reverse the electrodes in the power supply.
    4. Putting the gel in backwards in the apparatus.

Safety

  • Use nitrile gloves when handling acrylamide.
  • Dispose of acrylamide gels and trays as hazardous.

See here for detailed safety information.

References

  1. NuPAGE Technical Guide [NuPAGETechnicalGuide]
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