Difference between revisions of "Team:NU Kazakhstan/Notebook"
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<p> Document the dates you worked on your project.</p> | <p> Document the dates you worked on your project.</p> | ||
| + | <p>MAKING COMPETENT CELLS | ||
| + | 1. Inoculate a single colony into 5 mL LB in 50 mL falcon tube (taped on a loosed tap). | ||
| + | 2. Grow on 37°C with shaking for 130 rpm overnight. | ||
| + | 3. Use 1 mL to inoculate 100 mL to inoculate 100 mL of LB in 250 mL bottle the next morning. | ||
| + | 4. Shake 37°C for 1.5-3 hours. | ||
| + | 5. When OD is between 0.3-0.4 at 600 nm wavelength put cell on ice. | ||
| + | 6. Hold cells on ice for the 10 minutes. | ||
| + | 7. Collect cells by centrifugation for 3 min at maximum speed (4700 rpm). | ||
| + | 8. Decant supernatant and gently resuspend on 10 mL ice-cold 0.1 M CaCl2 (prepared in ddH2O). Treat them gently. | ||
| + | 9. Incubate on ice for 20 minutes. | ||
| + | 10. Centrifuge again at maximum speed (4700 rpm). | ||
| + | 11. Discard supernatant and gently resuspend in 5 mL cold 0.1 M CaCl2 (15% glycerol). | ||
| + | 12. Dispense into chilled microtubes, put on the dry ice. Perform this procedure very quickly. | ||
| + | 13. Freeze in -80°C. | ||
| + | TRANSFORMATION | ||
| + | 1. Start thawing the competent cells on ice. | ||
| + | 2. Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control. | ||
| + | 3. Add 1 - 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice. | ||
| + | 4. Add 1 µL of the RFP Control to your control transformation. | ||
| + | 5. Close tubes and incubate the cells on ice for 30 minutes. | ||
| + | 6. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. | ||
| + | 7. Incubate the cells on ice for 5 minutes. | ||
| + | 8. Add 200 μl of SOC media (make sure that the broth does not contain antibiotics and is not contaminated) to each transformation | ||
| + | 9. Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin. | ||
| + | 10. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony. | ||
| + | 11. For the control, label two petri dishes with LB agar (AMP). Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. | ||
| + | 12. Incubate the plates at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria. | ||
| + | 13. You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep. | ||
| + | 14. Count the colonies on the 20 μl control plate and calculate your competent cell efficiency. | ||
| + | LIGATION | ||
| + | 1. Add 2ul of digested plasmid backbone (25 ng) | ||
| + | 2. Add equimolar amount of EcoRI-HF SpeI digested fragment (< 3 ul) | ||
| + | 3. Add equimolar amount of XbaI PstI digested fragment (< 3 ul) | ||
| + | 4. Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase | ||
| + | 5. Add 0.5 ul T4 DNA ligase | ||
| + | 6. Add water to 10 ul | ||
| + | 7. Ligate 16C/30 min, heat kill 80C/20 min | ||
| + | 8. Transform with 1-2 ul of product | ||
| + | DNA EXTRACTION FROM CELLS (MINIPREP) | ||
| + | 1. Harvest. Centrifuge 1-5 mL of the overnight LB-culture (Use 1-2×104 E.coli cells for each sample). Remove all medium. Add 2 mL of ddH2O and centrifuge again. Remove all medium. | ||
| + | 2. Resuspend. Add 250 uL Resuspension Buffer (R3) with RNase A to the cell pellet and resuspend the pellet until it is homogenous. | ||
| + | 3. Lyse. Add 250 uL Lysis Buffer (L7). Mix gently by inverting the capped tube until the mixture is homogenous. Do not vortex. Incubate the tube at room temperature for 5 minutes. | ||
| + | 4. Precipitate. Add 350 uL Precipitation Buffer (N4). Mix immediately by inverting the tube, or for large pellets, vigorously shaking the tube, until the mixture is homogenous. Do not vortex. Centrifuge the lysate at >12,000 g for 10 minutes. | ||
| + | 5. Bind. Load the supernatant from step 4 onto a spin column in a 2-mL wash tube. Centrifuge the column at 12,000 g for 1 minute. Discard the flow-through and place the column back into the wash tube. | ||
| + | 6. Optional wash (Recommended for endA+ strains). Add 500 uL Wash Buffer (W10) with ethanol to the column. Incubate the column for 1 minute at room temperature. Centrifuge the column at 12,000 g for 1 minute. Discard the flow-through and place the column back into the wash tube. | ||
| + | 7. Wash and remove ethanol. Add 700 uL Wash Buffer (W9) with ethanol to the column. Centrifuge the column at 12,000 g for 1 minute. Discard the flow-through and place the column back into the wash tube. Centrifuge the column at 12,000 g for 1 minute. Discard the flow-through. | ||
| + | 8. Elute. Place the Spin Column in a clean 1.5-mL recovery tube. Add 75 uL of preheated TE Buffer (TE) to the center of the column. Incubate the column for 1 minute at room temperature. | ||
| + | 9. Recover. Centrifuge the column at 12,000 g for 2 minutes. The recovery tube contains the purified plasmid DNA at 4⁰C (short-term) or store DNA in aliquots at -20⁰C (long-term). | ||
| + | |||
| + | DNA EXTRACTION FROM GEL | ||
| + | Excising and dissolving the gel | ||
| + | 1. Equilibrate a water bath or heat block to 50⁰C. | ||
| + | 2. Excise a minimal area of gel containing the DNA fragment of interest. | ||
| + | 3. Weigh the gel slice containing the DNA fragment using a scale sensitive to 0.001 g. | ||
| + | 4. Add Gel Solubilization Buffer (L3) to the excised gel in the tube size indicated in the following table: | ||
| + | Gel Tube Buffer L3 Volume | ||
| + | ≤2% agarose 1.7-mL polypropylene 3:1 (i.e. 1.2 mL Buffer L3 : 400 mg gel piece) | ||
| + | >2% agarose 5-mL polypropylene 6:1 (i.e. 2.4 mL Buffer L3 : 400 mg gel piece) | ||
| + | 5. Place the tube with the gel slice and Buffer L3 into a 50⁰C water bath or heat block. Incubate the tube at 50⁰C for 10 minutes. Invert the tube every 3 minutes to mix and ensure gel dissolution. | ||
| + | 6. After the gel slice appears dissolved, incubate the tube for an additional 5 minutes. | ||
| + | 7. Optional: For optimal DNA yields, add 1 gel volume of isopropanol to the dissolved gel slice. Mix well. | ||
| + | 8. Purify the DNA using a centrifuge. | ||
| + | Purifying DNA using a centrifuge | ||
| + | 1. Load. Pipet the dissolved gel piece onto a Quick Gel Extraction Column inside a Wash Tube. Use 1 column per 400 mg of agarose gel. Note: the column reservoir capacity is 850 uL. | ||
| + | 2. Bind. Centrifuge the column at >12,000 g for 1 minute. Discard the flow-through and place the column into the Wash Tube. | ||
| + | 3. Wash. Add 50 uL Wash Buffer (W1) containing ethanol to the column. | ||
| + | 4. Remove Buffer. Centrifuge the column at >12,000 g for 1 minute. Discard the flow-through and place the column into the Wash Tube. | ||
| + | Repeat Steps 3 and 4. | ||
| + | 5. Remove Ethanol. Centrifuge the column at maximum speed for 3 minutes. Discard the flow-through. | ||
| + | 6. Elute. Place the column into a Recovery Tube. Add 50 uL Elution Buffer (E5) to the center of the column. Incubate the tube for 2 minutes at room temperature. | ||
| + | 7. Collect. Centrifuge the tube at >12,000 g for 5 minutes. | ||
| + | 8. Store. The elution tube contains the purified DNA. Store the purified DNA at 4⁰C for immediate use or at -20⁰C for long-term storage. | ||
| + | DNA ISOLATION FROM BACTERIA | ||
| + | 1. Pick an isolated bacterial colony and resuspend it in 1 mL of autoclaved water in a microfuge tube. | ||
| + | 2. Centrifuge for 1 minute at 10,000-12,000 rpm. Remove the supernatant. | ||
| + | 3. Add 200 uL of InstaGene matrix to the pellet and incubate at 56⁰c for 15-30 minutes. | ||
| + | Note: InstaGene matrix should be mixed at moderate speed on a magnetic stirrer to maintain the matrix in suspension. The pipet tip used should have a large bore, such as 1,000 uL pipet tip | ||
| + | 4. Vortex at high speed for 10 seconds. Place the tube in a 100⁰C heat block or boiling water bath for 8 minutes. | ||
| + | 5. Vortex at high speed for 10 seconds. Spin at 10,000-12,000 rpm for 2-3 minutes. | ||
| + | 6. Use 20 uL of the resulting supernatant per 50 uL PCR reaction. Store the remainder of the supernatant at -20⁰C. Repeat Step 5 when reusing the InstaGene preparation. | ||
| + | Note: It is important to store the prepared sample at -20⁰C. | ||
| + | Results and Discussion | ||
| + | ISOLATION OF S.MUTANS | ||
| + | The amplicon of the size 479 base pair was identified. On the picture of the gel below, the first column contains the ladder, the second one is the amplicon which was PCR-ed with the extension time of 1 minute, and the third one is the amplicon with the extension time of 14 seconds. The negative control with the Bacillus Subtilis extracted with Instagene matrix genome was performed. | ||
| + | |||
| + | Figure 1. Gel running of the S.mutans genome. | ||
| + | BLUE LIGHT SYSTEM | ||
| + | Restriction digest of the FixJ with the EcoRI and XbaI. Gel extraction of the cut FixJ. The concentration of the cut FixJ = 2.217 ng/ul. | ||
| + | |||
| + | Figure 2. Gel extraction of FixJ (the second column from the ladder with already cut piece) digested with the EcoRI and XbaI enzymes. | ||
| + | Restriction Digest of the pVeg with EcoRI and SpeI. Gel extraction of the cut pVeg. The concentration of the cut Pveg = 2.892 ng/ul | ||
| + | |||
| + | Figure 3. Gel extraction of pVeg (the second column from the ladder with already cut piece) digested with the EcoRI and SpeI enzymes. | ||
| + | The checking of the ligation of the Pveg+FixJ miniprep product by making sequential digest with SpeI (Sib enzyme) and EcoRI (fermentas). | ||
| + | As can be seen from the picture of the gel below, there are two distinct bands in the second row after the ladder. The upper one is the 2500 base pair DNA fragment. The following one is 2000 base pair DNA fragment. However, if Pveg+ FixJ ligation have worked there would be also an uncut plasmid after digestion of size 4500 base pair. So, it was decided to make a single digest procedure with EcoRI in order to check whether there would be a linearized plasmid of 4500 base pair size. | ||
| + | |||
| + | Figure 4. Gel running of the Pveg+ FixJ ligation product obtained as a result of a sequential digest with SpeI (Sib enzyme) and EcoRI (fermentas) | ||
| + | Single digest of the Pveg+FixJ with the EcoRI (Neb enzyme). | ||
| + | On the picture of the gel below, the first column is the ladder, the second one is the plasmid after ligation of Pveg+FixJ, the third one is the Pveg+FixJ plasmid cut with EcoRI. The results show that there is no linearized plasmid with the 4500 base pair after ligation. So ligation did not work. | ||
| + | |||
| + | Figure 5. Gel running of the Pveg+ FixJ ligation product obtained as a result of a single digest of the Pveg+FixJ with the EcoRI (Neb enzyme) | ||
| + | </p> | ||
<h5>What should this page have?</h5> | <h5>What should this page have?</h5> | ||
<ul> | <ul> | ||
Revision as of 19:41, 25 July 2015
Notebook
Document the dates you worked on your project.
MAKING COMPETENT CELLS 1. Inoculate a single colony into 5 mL LB in 50 mL falcon tube (taped on a loosed tap). 2. Grow on 37°C with shaking for 130 rpm overnight. 3. Use 1 mL to inoculate 100 mL to inoculate 100 mL of LB in 250 mL bottle the next morning. 4. Shake 37°C for 1.5-3 hours. 5. When OD is between 0.3-0.4 at 600 nm wavelength put cell on ice. 6. Hold cells on ice for the 10 minutes. 7. Collect cells by centrifugation for 3 min at maximum speed (4700 rpm). 8. Decant supernatant and gently resuspend on 10 mL ice-cold 0.1 M CaCl2 (prepared in ddH2O). Treat them gently. 9. Incubate on ice for 20 minutes. 10. Centrifuge again at maximum speed (4700 rpm). 11. Discard supernatant and gently resuspend in 5 mL cold 0.1 M CaCl2 (15% glycerol). 12. Dispense into chilled microtubes, put on the dry ice. Perform this procedure very quickly. 13. Freeze in -80°C. TRANSFORMATION 1. Start thawing the competent cells on ice. 2. Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control. 3. Add 1 - 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice. 4. Add 1 µL of the RFP Control to your control transformation. 5. Close tubes and incubate the cells on ice for 30 minutes. 6. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. 7. Incubate the cells on ice for 5 minutes. 8. Add 200 μl of SOC media (make sure that the broth does not contain antibiotics and is not contaminated) to each transformation 9. Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin. 10. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony. 11. For the control, label two petri dishes with LB agar (AMP). Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. 12. Incubate the plates at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria. 13. You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep. 14. Count the colonies on the 20 μl control plate and calculate your competent cell efficiency. LIGATION 1. Add 2ul of digested plasmid backbone (25 ng) 2. Add equimolar amount of EcoRI-HF SpeI digested fragment (< 3 ul) 3. Add equimolar amount of XbaI PstI digested fragment (< 3 ul) 4. Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase 5. Add 0.5 ul T4 DNA ligase 6. Add water to 10 ul 7. Ligate 16C/30 min, heat kill 80C/20 min 8. Transform with 1-2 ul of product DNA EXTRACTION FROM CELLS (MINIPREP) 1. Harvest. Centrifuge 1-5 mL of the overnight LB-culture (Use 1-2×104 E.coli cells for each sample). Remove all medium. Add 2 mL of ddH2O and centrifuge again. Remove all medium. 2. Resuspend. Add 250 uL Resuspension Buffer (R3) with RNase A to the cell pellet and resuspend the pellet until it is homogenous. 3. Lyse. Add 250 uL Lysis Buffer (L7). Mix gently by inverting the capped tube until the mixture is homogenous. Do not vortex. Incubate the tube at room temperature for 5 minutes. 4. Precipitate. Add 350 uL Precipitation Buffer (N4). Mix immediately by inverting the tube, or for large pellets, vigorously shaking the tube, until the mixture is homogenous. Do not vortex. Centrifuge the lysate at >12,000 g for 10 minutes. 5. Bind. Load the supernatant from step 4 onto a spin column in a 2-mL wash tube. Centrifuge the column at 12,000 g for 1 minute. Discard the flow-through and place the column back into the wash tube. 6. Optional wash (Recommended for endA+ strains). Add 500 uL Wash Buffer (W10) with ethanol to the column. Incubate the column for 1 minute at room temperature. Centrifuge the column at 12,000 g for 1 minute. Discard the flow-through and place the column back into the wash tube. 7. Wash and remove ethanol. Add 700 uL Wash Buffer (W9) with ethanol to the column. Centrifuge the column at 12,000 g for 1 minute. Discard the flow-through and place the column back into the wash tube. Centrifuge the column at 12,000 g for 1 minute. Discard the flow-through. 8. Elute. Place the Spin Column in a clean 1.5-mL recovery tube. Add 75 uL of preheated TE Buffer (TE) to the center of the column. Incubate the column for 1 minute at room temperature. 9. Recover. Centrifuge the column at 12,000 g for 2 minutes. The recovery tube contains the purified plasmid DNA at 4⁰C (short-term) or store DNA in aliquots at -20⁰C (long-term). DNA EXTRACTION FROM GEL Excising and dissolving the gel 1. Equilibrate a water bath or heat block to 50⁰C. 2. Excise a minimal area of gel containing the DNA fragment of interest. 3. Weigh the gel slice containing the DNA fragment using a scale sensitive to 0.001 g. 4. Add Gel Solubilization Buffer (L3) to the excised gel in the tube size indicated in the following table: Gel Tube Buffer L3 Volume ≤2% agarose 1.7-mL polypropylene 3:1 (i.e. 1.2 mL Buffer L3 : 400 mg gel piece) >2% agarose 5-mL polypropylene 6:1 (i.e. 2.4 mL Buffer L3 : 400 mg gel piece) 5. Place the tube with the gel slice and Buffer L3 into a 50⁰C water bath or heat block. Incubate the tube at 50⁰C for 10 minutes. Invert the tube every 3 minutes to mix and ensure gel dissolution. 6. After the gel slice appears dissolved, incubate the tube for an additional 5 minutes. 7. Optional: For optimal DNA yields, add 1 gel volume of isopropanol to the dissolved gel slice. Mix well. 8. Purify the DNA using a centrifuge. Purifying DNA using a centrifuge 1. Load. Pipet the dissolved gel piece onto a Quick Gel Extraction Column inside a Wash Tube. Use 1 column per 400 mg of agarose gel. Note: the column reservoir capacity is 850 uL. 2. Bind. Centrifuge the column at >12,000 g for 1 minute. Discard the flow-through and place the column into the Wash Tube. 3. Wash. Add 50 uL Wash Buffer (W1) containing ethanol to the column. 4. Remove Buffer. Centrifuge the column at >12,000 g for 1 minute. Discard the flow-through and place the column into the Wash Tube. Repeat Steps 3 and 4. 5. Remove Ethanol. Centrifuge the column at maximum speed for 3 minutes. Discard the flow-through. 6. Elute. Place the column into a Recovery Tube. Add 50 uL Elution Buffer (E5) to the center of the column. Incubate the tube for 2 minutes at room temperature. 7. Collect. Centrifuge the tube at >12,000 g for 5 minutes. 8. Store. The elution tube contains the purified DNA. Store the purified DNA at 4⁰C for immediate use or at -20⁰C for long-term storage. DNA ISOLATION FROM BACTERIA 1. Pick an isolated bacterial colony and resuspend it in 1 mL of autoclaved water in a microfuge tube. 2. Centrifuge for 1 minute at 10,000-12,000 rpm. Remove the supernatant. 3. Add 200 uL of InstaGene matrix to the pellet and incubate at 56⁰c for 15-30 minutes. Note: InstaGene matrix should be mixed at moderate speed on a magnetic stirrer to maintain the matrix in suspension. The pipet tip used should have a large bore, such as 1,000 uL pipet tip 4. Vortex at high speed for 10 seconds. Place the tube in a 100⁰C heat block or boiling water bath for 8 minutes. 5. Vortex at high speed for 10 seconds. Spin at 10,000-12,000 rpm for 2-3 minutes. 6. Use 20 uL of the resulting supernatant per 50 uL PCR reaction. Store the remainder of the supernatant at -20⁰C. Repeat Step 5 when reusing the InstaGene preparation. Note: It is important to store the prepared sample at -20⁰C. Results and Discussion ISOLATION OF S.MUTANS The amplicon of the size 479 base pair was identified. On the picture of the gel below, the first column contains the ladder, the second one is the amplicon which was PCR-ed with the extension time of 1 minute, and the third one is the amplicon with the extension time of 14 seconds. The negative control with the Bacillus Subtilis extracted with Instagene matrix genome was performed. Figure 1. Gel running of the S.mutans genome. BLUE LIGHT SYSTEM Restriction digest of the FixJ with the EcoRI and XbaI. Gel extraction of the cut FixJ. The concentration of the cut FixJ = 2.217 ng/ul. Figure 2. Gel extraction of FixJ (the second column from the ladder with already cut piece) digested with the EcoRI and XbaI enzymes. Restriction Digest of the pVeg with EcoRI and SpeI. Gel extraction of the cut pVeg. The concentration of the cut Pveg = 2.892 ng/ul Figure 3. Gel extraction of pVeg (the second column from the ladder with already cut piece) digested with the EcoRI and SpeI enzymes. The checking of the ligation of the Pveg+FixJ miniprep product by making sequential digest with SpeI (Sib enzyme) and EcoRI (fermentas). As can be seen from the picture of the gel below, there are two distinct bands in the second row after the ladder. The upper one is the 2500 base pair DNA fragment. The following one is 2000 base pair DNA fragment. However, if Pveg+ FixJ ligation have worked there would be also an uncut plasmid after digestion of size 4500 base pair. So, it was decided to make a single digest procedure with EcoRI in order to check whether there would be a linearized plasmid of 4500 base pair size. Figure 4. Gel running of the Pveg+ FixJ ligation product obtained as a result of a sequential digest with SpeI (Sib enzyme) and EcoRI (fermentas) Single digest of the Pveg+FixJ with the EcoRI (Neb enzyme). On the picture of the gel below, the first column is the ladder, the second one is the plasmid after ligation of Pveg+FixJ, the third one is the Pveg+FixJ plasmid cut with EcoRI. The results show that there is no linearized plasmid with the 4500 base pair after ligation. So ligation did not work. Figure 5. Gel running of the Pveg+ FixJ ligation product obtained as a result of a single digest of the Pveg+FixJ with the EcoRI (Neb enzyme)
What should this page have?
- Chronological notes of what your team is doing.
- Brief descriptions of daily important events.
- Pictures of your progress.
- Mention who participated in what task.
Inspiration
You can see what others teams have done to organize their notes: