July Notebook

7.9.14: Ligation

Background

• vector = plasmid
• insert = gene of interest = GFP in this case
• Synbiota does something weird to decimal points, so some numbers are represented with spaces and decimal points.

This is what we actually did

Calculate the appropriate molar ratios (see "Calculating Molar Ratios").

We decided to use an even higher ratio than 3:1 of gene of interest to plasmid.  We used 6 uL of the gene of interest and 4 uL of the plasmid to help increase the probability of combining the GFPs to the plasmids

We need to sum to 20 uL of solution total.

9 uL of deionized H20

4 uL of digested plasmid

6 uL of PCR purified GFP

2 uL of 2x ligation buffer

1 uL of ligase

~20 uL total (actually 21 uL)

This is from the NEB website: https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202

 COMPONENT 20 μl REACTION 10X T4 DNA Ligase Buffer* 2 μl Vector DNA (4 kb) 50 ng (0 . 020 pmol) Insert DNA (1 kb) 37.5 ng (0 . 060 pmol) Nuclease-free water to 20 μl T4 DNA Ligase 1 μl

* The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.

Procedure

This is what we actually did:

1. Combine the materials in a PCR tube in THIS ORDER:
1. H2O
2. buffer
3. DNA (plasmid and GFP, the order does not matter)
4. enzyme (ligase)
2. Mix with pipettor.
3. Incubate at 16C in the PCR machine. Actually was at 16C for 24 minutes then room temperature for about 23.5 hours.
4. Put on ice in the freezer.

This is from the NEB website: https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202

1. Set up the following reaction in a microcentrifuge tube on ice.
2. (T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.) Use NEBioCalculator to calculate molar ratios.
3. Gently mix the reaction by pipetting up and down and microfuge briefly.
4. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
5. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours(alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
6. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.

7.29.14: PCR Purification and Ligation

PCR Purification:

Key for the contents of each tube:

• Tube A = OFP
• Tube B = YFP
• Tube C = RFP
• Tube D = CFP
• Tube BB = Backbone

We used Life Technologies Purelink Invitrogen PCR purification protocol. (See below and attached protocols in pdf format downloaded from Life technologies website on 7/31/14).

Catalog numbers K3100-01 and K3100-02

Publication Part Number 7015021 MAN0004375 Revision Date 14 September 2011

Procedure for Purifying PCR Products

1. Combine. Add 4 volumes of Binding Buffer B2 or B3 with isopropanol (see preceding table) to 1 volume of a PCR sample (50–100 μL). Mix well.

2. Load. Pipet the sample into a PureLink® Spin Column in a Collection Tube. Centrifuge the column at >10,000 × g for 1 minute. Discard the flow-through.

3. Wash. Re-insert the column into the Collection Tube and add 650 µL Wash. Buffer (W1) with ethanol. Centrifuge the column at >10,000 × g for 1 minute. Discard the flow-through and place the column in the same Collection Tube. Centrifuge the column at maximum speed for 2–3 minutes.

4. Elute. Place the column into a clean 1.7-mL Elution Tube (supplied with the kit). Add 50 µL Elution Buffer to the center of the column. Incubate the column at room temperature for 1 minute. Centrifuge the column at maximum speed for 2 minutes. The elution tube contains the purified PCR product. Store the purified DNA at 4°C for immediate use or at −20°C for long-term storage.

We made the following changes to the above protocol:

1. We started with 20 µL of digest.
2. We used Elution buffer only on tube A.
3. We used deionized Water as buffer for tube B, C, D. The reason for this change is that we felt there might be a conflict between elution buffer and ligation buffer used during ligation process. (For more details ask Julie)

Ligation:

14 µL of digested insert (from PCR purification) (Old inserts PCR purified were digested with ecoR1/A+I) (As mentioned above: EB used for A tube. dH2O used for B-D)

3 µL BB (1:100 dilution in H20) (BB=pSBIC3 Ecoli/Ps+I digested on 7-10-14 + PCR purified)

2 µL buffer

1 µL Ligase

20 µL total

7.31.14: Transformation of ligation products (Backbone, RFP, GFP, YFP, CFP) into Invitrogen One-Shot Top10 E. coli cells

Background

We followed the protocol for Invitrogen Life Technologies One Shot® TOP10 Competent Cells (see attached pdf downloaded on 7/31/14 from invitrogen and excerpt below):

Transform chemically competent cells: Chemical transformation procedure

1. Centrifuge the vial(s) containing the ligation reaction(s) briefly and place on ice.

2. Thaw, on ice, one 50 μL vial of One Shot® cells for each ligation/transformation.

3. Pipet 1–5 μL of each ligation reaction directly into the vial of competent cells and mix by tapping gently. Do not mix by pipetting up and down. The remaining ligation mixture(s) can be stored at −20°C.

4. Incubate the vial(s) on ice for 30 minutes.

5. Incubate for exactly 30 seconds in the 42°C water bath. Do not mix or shake.

6. Remove vial(s) from the 42°C bath and place them on ice.

7. Add 250 μL of pre-warmed S.O.C medium to each vial. S.O.C is a rich medium; sterile technique must be practiced to avoid contamination.

8. Place the vial(s) in a microcentrifuge rack on its side and secure with tape to avoid loss of the vial(s). Shake the vial(s) at 37°C for exactly 1 hour at 225 rpm in a shaking incubator.

9. Spread 20–200 μL from each transformation vial on separate, labeled LB agar plates. The remaining transformation mix may be stored at 4°C and plated out the next day, if desired.

10. Invert the plate(s) and incubate at 37°C overnight.

11. Select colonies and analyze by plasmid isolation, PCR, or sequencing.

We transformed the following ligation products:

• Tube A = OFP
• Tube B = YFP
• Tube C = RFP
• Tube BB = Backbone
• Did not transform Tube D = CFP (Only had 4 vials of competent cells so did not transform CFP)

For step 3, we pipetted 5 μL of each ligation reaction directly into the vial of competent cells.

For step 7, we did not have SOC media so we substituted LB for SOC media.

For step 9 we added the following procedure:

• Spin down 3 mins at 4,000 RPM
• Aspirate and discard 150 microliters
• Resuspend in remaining ~100 microliters
• Plate to LB+Chl

Results

No colonies on any plates. Our hypothesis for the poor result is that we used a 1:100 dilution of backbone. It might also be that the cells we were using we not competent.