Katherine K. Jacobs: Notebook

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(Ligation Procedure:)
Current revision (16:45, 12 March 2008) (view source)
(Ligation Procedure:)
 
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*A helpful equation for determining the correct amount of insert:
*A helpful equation for determining the correct amount of insert:
-
*''Insert mass in ng = 3 x [Insert Length (bp)/Vector Length (bp)] x Vector mass in ng''
+
*''Insert mass in ng = 3 x [Insert Length (bp)/Vector Length (bp)] x Vector mass in ng''
-
+
-
+
*      5. Place the cap on the tube and pulse the tubes in a microfuge or a vortex.
*      5. Place the cap on the tube and pulse the tubes in a microfuge or a vortex.
*      6. If using the reaction immediately after, incubate the reaction at room temperature for 2-
*      6. If using the reaction immediately after, incubate the reaction at room temperature for 2-
  24 hours.  If the reaction will be used at a later date, incubate at -20 deg C until it is         
  24 hours.  If the reaction will be used at a later date, incubate at -20 deg C until it is         
               ready for use.
               ready for use.

Current revision

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Contents

Lab Introduction January 30, 2008

3:30 PM Pipette Use

  • Sterilization of the pipette with ethanol
  • Pipette #1: 2 microliters blue solution and 5 microliters water
  • Set pipette to 7 microliters to test accuracy of previous measurements
  • Pipette #2: 20 microliters blue solution and 50 microliters water

Making Solutions:

  • Solution #1: KCl Potassium Chloride 0.3 M
  • Solution #2: MgCl2 Magnesium Chlroide 0.5 M
  • Solution #3: NaCl Sodium Chloride 1 M
  • Pour .5 of water amount into container, add salt then add the rest of the water to assist in mixture of two substances
  • Preparation of Magnesium Chloride Solution:

(203.31 grams/ mole) x (0.5 moles/Liter) x (0.1 Liter) = 10.1655 grams/ 100 mL

  • Preparation of Potassium Chloride Solution:

(74.56 grams/mole) x (0.3 moles/Liter) x (0.1 Liter) = 2.2368 grams/ 100 mL

4: 30 PM Autoclave:

  • Mix 25 grams of LB mixture and 15 grams of Bactoagar to a 2 L flask and autoclave for 20 minutes
  • Once autoclave was complete, the tape on the flask did not turn black indicating that the mixture was not successfully sterilized.
  • Suggestion for solving problem – Autoclave for 40 minutes and set to Sterilize

5:30 PM Making Cell Cultures:

  • LB with Ampicillin contains nutrients for the cells to grow
  • Keep the culture tubes (with a loose lid) sterile by closing the bag after removing tubes
  • The culture should get cloudy while the control should remain clear
  • Sterilize the pipette with ethanol
  • 2 mL cultures are optimal
  • Fill both tubes with the LB first
  • Sterilize the loop by placing in ethanol and then flaming
  • When removing a colony from a plate, mark on the underside of the plate with a marker which colony has been taken
  • Incubate the culture overnight at 37 degrees C in a shaking water bath

Transformation of E. coli with Recombinant DNA February 6, 2008

  • Competent E. coli cells are transformed with commercial competent cells (JM109 E. coli strain, Promega, catalog #: L2001) ligated with DNA from the iGEM plates.
  • Registry of BioBrick parts: parts.MIT.edu
  • Positive controls: BBa_I13522 and BBa_J45220

Group 1 (Adam Crego and Katherine):

  • BioBricks: a) BBa_R0011 b) BBa_J45219
  • Katherine: BBa_R0011 Adam: BBa_J45219
  • BBa_R0011: Promoter (lacI regulated, lambda pL hybrid) ON in strains without lacI, OFF in strains lacq, medium in strains with lacI. 55 base pairs in length. PHYSICAL DNA: Well- 7M Plate- 1 Plasmid- pSB1A2.
  • To avoid the problem of running out of DNA, Glycerol Stocks (freezing the DNA) can be done after amplification and transformation.
  • Labeling is done by writing Control or Part Name, initials, and date of experiment.

Transformation Procedure

  1. 2:50 PM: BBa_R0011 is taken out of the well with 9 microliters of distilled water.
  2. One centrifuge tube labeled Control/KKJ/2-6-08 and another labeled R0011/KKJ/2-6-08. Place both in ice. The control in this experiment is a negative control because no DNA is added to the competent cells.
  3. Obtain 100 microliters of competent cells for each centrifuge tube. Competent cells must thaw before being used.
  4. 3:35 PM: 4 microliters of BBa_R0011 added to competent cells and left in ice for 20 minutes.
  5. 3:55 PM: Both control and DNA centrifuge tubes are placed in a 42 degree C hot bath for 60 seconds.
  6. Both tubes are placed back in the ice for two minutes.
  7. Add 900 microliters of ice cold antibiotic free LB to both centrifuge tubes.
  8. 4:10 PM: Cells are incubated in 37 degree C for 60-90 minutes.
  9. 5:20 PM: Three solutions are plated on LB ampicillin resistant plates. Spreader dipped in ethanol and passed through a flame to ensure sterilization before plating. The three plates contained: Plate 1) 100 microliters of the control Plate 2) 100 microliters of LB/cells (1:1 ratio) Plate 3) Dilution of 99 microliters water and 1 microliter of LB/cells (100:1 ratio)
  10. Plates are incubated upside down for 24 hours in 37 degrees C.
  • February 7, 2008 6:00 PM: Plates were checked for growth but none exhibited any colony growth. This could be due to the commercially bought competent cells or the temperature of the overnight incubation. The temperature varied from 37 degrees C by a few degrees.

Plasmid Miniprep February 13, 2008

  • Since the transformation on February 6, 2008 was not succesful, glycerol stocks will be used for the miniprep.
  • Parts used: 1) E0241 - PoPs to GFP converter, RBS: B0032, GFP: E0040, Terminator: B0016 2) R0010 - promoter lacI regulator 3) C0040 - tetracycline repressor from Tn10 transposon + LVA (degradation tag) 4) R0040 - promoter (TetR -)
  • Cultures were made at 12:00 AM on February 13, 2008 and will be used 14-15 hours after at 2:30 PM February 13, 2008. It is important not to allow the cultures to incubate for long periods of time (days) because the cells can start to lyse.
  • Control shows no cloudiness while other cell cultures are cloudy at the bottom of the test tube.

Miniprep Prodedure

  • The purpose of this procedure is to extract plasmid DNA and separate it from chromosal DNA in a cell. All DNA (chromosal and plasmid) is denatured in the first step. Plasmid DNA, unlike chromosomal DNA, forms linked rings upon denaturation. Next, DNA is renatured with the use of buffers. Chromosomal DNA remains denatured because of the inefficiency of re-ligation of multiple pieces and strands of DNA. The plasmid DNA renatures and is thereby, separated from chromosomal DNA. Two rounds of the glycerol stock, C0040, were centrifuged to obtain as much DNA as possible before starting the miniprep.
  • Before starting the miniprep, the following steps should be completed with a new miniprep kit.
  1. Add RNase to Buffer P1 and store in the refrigerator.
  2. Add ethanol to Buffer PE.
  3. Check Buffers P2 and P3 for salt precipitation and redissolve at 37 degrees C is neccessary.
  • After preliminary steps have been done the miniprep is performed with the following steps:
  1. 2:30 PM: Resuspend the pelleted bacterial cells in 250 microliters Buffer P1 and put in a centrifuge tube.
  2. Add 250 microliters Buffer P2 and mix, invert 4-6 times. Solutions containing LyseBlue will turn blue.
  3. Add 350 microliters N3 and IMMEDIATELY invert 4-6 times. Solutions with LyseBlue will turn colorless.
  4. Centrifuge for 10 minutes at 13,000 rpm.
  5. Apply supernatant to QIAprep spin by decanting or pipetting.
  6. Centrifuge for 30-60 seconds at 13,000 rpm. Discard flow through.
  7. Wash QIAprep spin column. Add .5 mL PB. Centrifuge 30-60 seconds.
  8. Wash QIAprep spin column by adding .75 mL PE. Centrifuge 30-60 seconds.
  9. Dsicard flow through. Centrifuge 1 minute.
  10. To elute DNA, place QIAprep column in clean 1.5 mL centrifuge tube. Add 50 microliters EB or water to center of each QIAprep spin column, making sure to pipette EB right onto the cotton plug. Let stand for 1 minutes. Centrifuge for 1 minute.
  • Using the spectrometer, the DNA concentration is taken. A concentration of 20 ng/microliter is an ideal. C0040 DNA concentration = 12.2 ng/microliters.

Ethanol Precipitation February 13, 2008

  • Ethanol Precipitation is used to purify small fragments of DNA and increase DNA concentration. Since only 10 microliters of DNA can be placed in Gel Electrophoresis wells, the amount must be extremely concentrated.
  1. 4:15 PM: Add 1) 3 volumes of COLD 100% EtOH (50 microliters X 3 = 150 microliters) 2) 1/10 3M Sodium Acetate (20 microliters) 3) 1 microliter GlycoBlue
  2. 4:35 PM: Incubate for 1 hour at -80 deg. C
  3. 5:40 PM: Centrifuge 30 minutes at 0 deg. C. A blue pellet should be visible once finished centrifuging. Pellets will dissolve if in room temperature too long before performing next step!
  4. Remove supernatant.
  5. Air dry.
  6. Resuspend in water.

Gel Electrophoresis February 13, 2008

  • Gel Electrophoresis is primarily used to determine the length of a fragment of DNA. DNA Ladders are run beside experimental fragments as points of reference. A DNA Ladder contains all different lengths of DNA that when run out on a gel, separate to reveal the number of base pairs in each gene.
  1. Prepare the casting gel: 1% agarose gel - 1) 0.5 g agarose 2) 50 mL TBE + SYBR safe.
  2. Gel must be submerged completely with 300-500 mL (large gel tray) .5X TBE + SYBR safe buffer.
  3. Put the gel, WITHOUT the lid, in the microwave on HIGH for 1 minute.
  4. Cool the bottle of gel under water.
  5. Pour into the castings with rubber stoppers.
  6. The gel takes approximately 20 minutes to set. Once gel is set make sure to remove the rubber stopprs.
  7. DNA has a negative charge. The wells of the gel are aligned with on the negative side (Cathode). DNA will move away from the Cathode to the Anode (+).
  • The gel acts as a sieve for fragements as small as 100 base pairs and as large as 50,000 base pairs. 0.3% large fragments - 2% large fragments.
  1. When complete, pour TBE buffer back into a container. The buffer can be used up to 8 times.

Restriction Digest February 20, 2008

  • R0040 will be used instead of C0040 from the Plasmid Miniprep.
  • Restriction digest, also known as DNA fragmentation, is a method to cut DNA into smaller fragments of interest with restriction enzymes. In our experiment, we performed double digests which involves cutting the vector and insert DNA with two different restriction enzymes. XbaI and PstI restriction enzymes are used to cut the insert, in this case Green Fluorescent Protein (GFP). SpeI and PstI are used to cut the plasmid (ROO40 and R0010). GFP is designated as the insert because it is much larger (approximately 700 bp) than R0040 or R0010. If R0040 or R0010 were to be used as inserts, they would not show up clearly during the gel electrophoresis. It is important to use different restriction enzymes to ensure correct insert orientation during ligation with the vector. Things to take into account when performing a restriction digest: (1) Buffer compatibility: When performing a single digest, a buffer with a low salt concentration should be used first followed by a buffer with a higher salt concentration, (2) Depending on the amount of DNA, volumes of buffer and digest must be altered, (3) When performing a restriction digest on a PCR product, allow the reaction to run for 24 hours because PCR works with linear pieces of DNA. With plasmid DNA, the reaction needs to run for 2-6 hours.
  • All products are from Promega.

http://parts2.mit.edu/wiki/index.php/Image:StandardAssemblyTwoIntoOne.png

Plasmid Double Digest

  • Performed on R0010 and R0040. The neccesary components must be added in the following order:
  1. 5 microliters BSA (Bovine Serum Albumin)
  2. 5 microliters Buffer B
  3. 21 microliters DNA (200 ng - 1 microliter)
  4. 1.5 microliter SpeI
  5. 1.5 microliters PstI
  • Total = 34 microliters
  • BSA and Buffer B simulate the native environment of the restriction enzymes so they can work as efficiently as possible.

Vector Double Digest

  • Performed on E0241.
  1. 5 microliters BSA
  2. 5 microliters Buffer H
  3. 21 microliters DNA
  4. 1.5 microliters XbaI
  5. 1.5 microliters PstI
  • Total = 34 microliters
  • 2:35 PM Both double digests are incubated for two hours at 37 deg. C

Gel Electrophoresis February 20, 2008

  • 4:30 PM The double digests are removed from incubation.
  • Components of a DNA ladder: (1) 1 microliter 1 kilobase ladder (2) 2 microliters BlueJuice Dye (3) 7 microliters water
  1. Add 2 microliters of loading dye to the reaction so the DNA can be loaded into the wells of the gel more easily. The DNA solution will turn blue.
  2. Add 17 microliters of plasmid DNA to needed number of wells (34 microliters / 17 microliters = 2 wells in gel)
  3. 5:10 PM Run the gel at 100 Volts
  4. 6:05 PM Stop running gel
  • Long wave or short wave UV light can be used to look at a gel. Long wave UV is preferable to short wave UV because it prevents adjacent thymidine binding which often happens with short wave UV. Adjacent thymidine binding prevents cloning because the ribosome will stop short. Make sure to use a loading gel that is easy to discern (ie. purple instead of white) when using long wave UV.
  1. 6:20 PM Gel bands are cut, Weight of centrifuge tube without DNA: 1.0183 g Centrifuge tube with cut DNA bands: 1.3138 g Mass of gel: .2955 grams.

Gel Extraction February 27, 2008

  1. Excise the DNA fragment from the gel after performing gel electrophoresis.
  2. Weigh the cut pieces of gel and add 3 Volumes of Buffer QG to 1 Volume of gel (1 mg is approximately 1 microliter).
  • .2955 grams (mass of gel cut from gel) x 3 = (.8865 grams) x 1000 mg = 886.5 mg = 886.5 microliters (volume of Buffer QG added)
  1. 2:50 PM Incubate in 50 deg C for 10 minutes to dissolve the gel completely. Every 2-3 minutes during incubation, mix contents by vortexing.

The contents of the tube should be yellow after incubation.

  1. 3:00 PM Add 1 gel Volume of isopropanol. The addition of isopropanol will increase the yield of the fragements. It is used for fragements smaller than 500 base pairs of greater than 4 kb. It is optional for fragments in between 500 bp and 4 kb.
  2. Place a QIAquick spin column in a provided 2 mL collection tube.
  3. To bind the DNA, apply sample to QIAquick column and centrifuge for 60 seconds. The maximum volume for the spin column is 800 microliters.
  4. Discard flow through. Place QIAquick column into the same collection tube.
  5. Recommended step: Add .5 mL Buffer QG and centrifuge for 60 seconds.
  6. To wash the DNA, add .75 mL Buffer PE and centrifuge for 60 seconds.
  7. Discard flow through. Centrifuge for 60 seconds at 13,000 rpm.
  8. Place column into a clean 1.5 centrifuge tube.
  9. Add 34 microliters Buffer EB or water to center of the QIAquick membrane. Centrifuge for 60 seconds.
  10. Add loading gel if a gel analysis will be performed after gel extraction.

Calf CIP - Calf Intestinal Alkaline Phosphatase February 27, 2008

  • This procedure is only done to the vector plasmid. Calf CIP hydrolyzes the 5 prime end of the vector (phosphate group) so it cannot religate with itself.
  1. Elute gel extract in 30 microliters 10 mM Tris-HCl Buffer (Buffer EB)
  2. CIAP 10x Reaction Buffer and CIAP enzyme should thaw on ice until used. Add 4 microliters CIAP 10x Reaction Buffer and 1 microliter CIAP enzyme to the 34 microliters from the gel extraction. The total volume should be 39 microliters.
  3. 4:10 PM Incubate in 37 deg C for 30 minutes.
  4. 4:30 PM Add 1 microliter CIAP enzyme. Incubate in 37 deg C for 30 minutes.
  • An Ethanol Precipitation is performed on the vector after the Calf CIP procedure and on the insert after Gel Extraction. The Ethanol Precipitate is usually placed in -80 deg C overnight OR for 1 hour coupled with a 30 minute centrifuge at 4 deg C.

Ligation Reaction March 5, 2008

  • After incubation of the ethanol precipitate, resuspend the pellet in 3 microliters double distilled (dd) water before performing the ligation reaction.

Ligation Reaction Procedure

  1. Thaw T4 DNA ligase and 10x Buffer on ice.
  2. In a sterile tube add (in this specific order): 1. 8 microliters dd water 2. 1 microliter vector (R0040) 3. 3 microliters insert (E0241) 4. 1.5 microliter 10x Ligase Buffer 5. 1.5 microliter T4 DNA ligase
  3. 2:45 PM 2 Hour Benchtop Ligation (products are kept at room temperature between 2-24 hours). When performing a short incubation, the concentration of ligase should be increased. Normally 1 microliter of T4 DNA ligase is used. Here we are using 1.5 microliters. If not proceeding onto transformation after the incubation period is complete, the ligation reaction can be stopped by freezing at -20 deg C.
  • It is important to note here the 3:1 ratio used when combining the vector and insert for the ligation reaction. The ratio is in terms of molarity NOT volume. Depending on the size of the insert, the volume of insert used will vary. With a very small insert, the ratio of insert to vector might increase to around 10:1.
  • The following equation can be used: Insert mass in nanograms = 3 x [Insert length (bp) / Vector length (bp)] x Vector Mass in nanograms

Transformation Procedure

  1. Label Eppendorf Tubes: - (control), +, Part
  2. Thaw frozen competent cells on ice until just thawed. Do not let them get too warm.
  3. Gently mix thawed cells by flicking tube. DO NOT TITURATE.
  4. Add 100 microliters of competent cells to each tube. Add the 15 microliters of DNA from the ligation reaction to the "Part" test tube. Normally only 5 microliters is added but since we are not using the ligation reaction product for any other procedure we can use it all for the transformation. Do not put any DNA into the - control tube.
  5. IMMEDIATELY ice for 10 minutes
  6. Heat shock at 42 deg C for 45-60 seconds
  7. Ice 2 minutes
  8. Add 100 microliters LB to both tubes
  9. Ask "What is the resistance?" If the antibotic resistance is bacteriostatic (stopping growth), then the reaction does not need to be incubated for 90 minutes. If the antibiotic resistance is bacteriocidal (killing cells) then the reaction needs to be incubated so that the cells will develop resistance.
  10. Plate all 200 microliters from each tube onto their respective plates (-, +, Part)
  11. Incubate in 37 deg C overnight.
  • March 6, 2008 11:00 AM Plates checked for any colonies. No colonies detected on the control or Part plate.

March 7, 2008

  • 6:30 PM Cultures were made from the plates of Aaron's yeast and the parts from MIT. One colony was selected and placed in either LB + Kan or LB + Amp depending on its resistance. All parts were incubated in 37 deg C for 12-16 hours at 250 rpm.

March 8, 2008

  • 10:45 AM Rima started to make Glycerol Stocks with the cultures from the night before. 50 microliters of the part and 50 microliters of Glycerol were added and placed in the -80 deg C freezer.
  • 11:30 AM: Rima and started the miniprep for all parts. A miniprep is needed when ligating pieces together and putting them into a larger piece. The miniprep was finished at 3:00 PM and the parts were left in the refrigerator.

March 9, 2008

  • 6:30 PM Nanodrops were performed on each part.
  • 30 microliters were used in each nanodrop:
  1. pSB2K3 - E2020: 103.6
  2. pSB2K3 - E2030: 136.9
  3. pSB2K3 - E2050: 69.3
  4. pSB2K3 - E2060: 57.7
  5. J63010 - J63002: 63.9
  6. J63010 - J63003: 32.2
  7. J63010 - J63006: 160.3
  8. J63010 - J63007: 41
  9. J63010 - J63008: 22.1
  10. p1B1A2 - B0034: 14.2
  11. J01002 - J23100: 82
  12. pSB1A2 - J31002: 9.2
  13. pSB4A5 - 152001: 11.1
  • Aaron's Yeast Cultures:
  1. pRS306 Colony 1: 286.8
  2. pRS306 Colony 2: 259.7
  3. pRS305 Colony 1: 265.4
  4. pRS305 Colony 2: 264.1
  5. p416GPD Colony 1: 239
  6. p416GPD Colony 2: 197.7
  7. p415GPD Colony 1: 73.8
  8. p415GPD Colony 2: 103

Ligation Procedure:

  • Sources: Laboratory Experiments in Microbiology (Johnson and Case) and DNA Science (Micklos and Freyer
  • 1. Label a clean centrifuge tube “LIG”
  • 2. If performing a ligation after a restriction digest, heat the tubes from the restriction digest in 65 degrees C for 10 minutes.
  • 3. Thaw T4 DNA ligase and 10x Buffer on ice.
  • 4. Add the following reagents to the LIG tube:

1. 3 microliters water 2. 1 microliter vector 3. *3 microliter insert 4. 1.5 microliter Ligase Buffer 5. 1 microliter T4 DNA

  • The amount of vector and plasmid added will differ between each experiment based on the size of the insert. The idea ratio of vector to plasmid is 1:3 in terms of molarity and NOT VOLUME. If a short insert is being used, a larger volume of it will need to be used to ensure maximum ligation.

*A helpful equation for determining the correct amount of insert:

  • Insert mass in ng = 3 x [Insert Length (bp)/Vector Length (bp)] x Vector mass in ng
  • 5. Place the cap on the tube and pulse the tubes in a microfuge or a vortex.
  • 6. If using the reaction immediately after, incubate the reaction at room temperature for 2-

24 hours. If the reaction will be used at a later date, incubate at -20 deg C until it is

             ready for use.
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