GFP Fusion Cloning (Part IIIB): Gel to Check Recovery of Cloning DNAs MATERIALS:
ID: 210780 • Letter: G
Question
GFP Fusion Cloning (Part IIIB): Gel to Check Recovery of Cloning DNAs
MATERIALS:
1) numbered gel box with tray, comb and cover with wires
2) sterile 1.5ml microtubes
3) Pipetman-20 and sterile 200µl pipet tips
REAGENTS:
1) Running buffer (TBE): (89 mM Tris, 89mM Boric acid, 2mM EDTA) with 0.1 g/ml Ethidium Bromide
2) Hot molten 0.8% agarose in TBE with 0.1 ug/ml Ethidium Bromide
3) Loading buffer (40% glycerol, 0.25% bromophenol blue, 0.25% xylene cyanol)
4) 1Kb DNA ladder
PROCEDURE:
Caution: Put gloves on before you handle any of the gel apparatus as there may be traces of the mutagen ethidium bromide. When finished, remove these gloves and leave them by the gel apparatus before handling notebooks, micropipetors etc. so you do not contaminate them.
1. Before you handle any of the apparatus, notice that the box, which is used for the gel already, has buffer in it. It is supposed to be this way, so do not pour out the buffer. Also, this buffer contains ethidium bromide so do not get it on your hands.
NOTE: All the parts of the gel box that you are using should have the same number. DO NOT USE PARTS, WHICH HAVE DIFFERENT NUMBERS!
2. Wearing gloves, bring the gel tray in the plastic container to the gel casting area (do not carry the gel tray without the container as it may drip ethidium bromide buffer). Insert the gel tray into the gel caster (on table with agarose) so that the orange colored rubber seals are sealing against the sides of the gel caster (be careful not to roll the rubber seal out of it's groove otherwise the gel will leak out).
3. Place the gel comb into the slots closest to the end of the gel tray. Using a graduated cylinder, measure 45ml of molten agarose and pour it into the gel tray.
CAUTION: The agarose contains ethidium bromide. WEAR GLOVES while handling. Do not get the agarose on your skin or clothing. If contact does occur wash skin or clothing thoroughly with soap and water. Report any spills to the instructor!
Remove your gel gloves by pulling on the fingers so that they do not invert (so you can use the gloves again). Leave the gel gloves on the gel apparatus tray.
Allow a minimum of 30 minutes for the gel to completely solidify.
4. Spin the five tubes (VC, CTV, UTV, IC, Insert) containing your cloning DNAs in a microcentrifuge for a second or two to get all the material into the bottom of the tubes. Check that you have at least 70 µl of the CTV, UTV, and Insert. If you are unsure ask your Instructor.
5. If you have the minimum volumes, obtain (6) 1.5ml microtubes for samples and label them 1S to 6S. Add 1 µl of tracking dye to the 1S, 2S, 3S, 4S and 5S tubes. Add 2 µl of tracking dye to the 6S tube. Prepare a sample of each of the DNAs listed below by mixing each sample with the dye in the appropriately labeled sample tube (use a new micropipet tip for each sample).
tube #1S 5 µl of VC (Vector Control)
tube #2S 5 µl of CTV (CIP Treated Vector)
tube #3S 5 µl of UTV (UnTreated Vector)
tube #4S 5 µl of IC (Insert Control)
tube #5S 5 µl of Insert
tube #6S 10 µl of 1Kb DNA ladder
6. When your gel is ready, load the samples in the following manner:
Lane 1: empty
Lane 2: empty
Lane 3: 6 µl of 6S (1 Kb DNA ladder)
Lane 4: 6 µl of 1S (VC: Vector Control)
Lane 5: 6 µl of 2S (CTV: CIP Treated Vector)
Lane 6: 6 µl of 3S (UTV: UnTreated vector)
Lane 7: 6 µl of 4S (IC: Insert Control)
Lane 8: 6 µl of 5S (Insert)
Lane 9: 6 µl of 6S (1 Kb ladder)
Lane 10: empty
Lane 11: empty
Lane 12: empty
After loading, all "S" tubes, the VC and IC tubes can be disposed of. DO NOT THROW AWAY the CTV (CIP Treated Vector), UTV (UnTreated Vector), and Insert tubes!!!!!
7. Run the gel at 100 volts until the first dye front is 1/2 the way down the gel (approximately 60 to 90 minutes).
Note: while waiting, continue with GFP Fusion Cloning (Part IIIC): Determination of Plasmid DNA Concentration by Fluorometric Photometry
8. When the dye front has run 1/2 down the gel, turn off the power supply, put on gel gloves, remove the gel and gel tray from the gel box, put them into the plastic container containing a paper towel and bring them up to the front desk (DO NOT CARRY THE GEL WITH YOUR HANDS!!!).
9. Observe your gel on the U.V. box and photograph it. Do Not Dispose of the gel until you have your picture. Make sure to label the photograph: "GFP Fusion Cloning DNA Recovery".
10. Once you have your picture, in consultation with your instructor, decide if your restrictions are complete, incomplete or are not restricted. Also, decide if you have recovered enough of your DNAs to continue with the ligation procedure. If you have not recovered your DNAs then consult with your instructor.
11. Clean up: dispose of the gel in the Lab Waste (NOT THE SINK!). Wipe out the gel tray, the plastic container, make sure the comb is clean and the cover is on the gel box. Dispose of the gel gloves. DO NOT WASH THE GEL TRAY IN THE SINK AS YOU WILL CONTAMINATE THE HANDLES OF THE FAUCET WITH ETHIDIUM BROMIDE!!! DO NOT POUR THE BUFFER OUT OF THE GEL UNIT!
That is the lab , the question are:
•Describe your results:
For each fragment (insert and vector):
-did you recover the fragment after purification? If not, what do you think could have happened?
-are the fragments the expected sizes?
Explanation / Answer
Yes insert fragment and untreated and treated vectors are recovered, but in less concentration.
Vector is of high molecular weight and fragment to be inserted is of low molecular weight.
Both the vectors and insert are visible as faint band than the rest indicating less concentration.
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