National Centre for Biotechnology Education | Protocols

	The Lambda DNA protocol
	The Transformer protocol
	Illuminating DNA
	In a jam and out of juice
	Practical fermentation
	Practical biotechnology
	Nature's dice
	Protein power!
	Plant tissue culture
	Unpublished protocols

Formerly of the Centre for Studies in Science and Mathematics Education
The University of Leeds


	PLEASE NOTE		DOWNLOAD
	PLEASE NOTE

The following set of instructions assumes that you have downloaded the two files: uncut and master and copied them onto a disc. The files are all plain text files ending with the suffix '.doc'. Please configure your Web browser to download files with this suffix as word processor documents. The following instructions were written for use with Microsoft Word; other word processors can be used in a similar way.

A third file - simulate - is a copy of these instructions which can be modified to suit other word processors or different teaching needs.

	Uncut DOWNLOAD
	Master DOWNLOAD
	Simulate DOWNLOAD


	INSTRUCTIONS


	You are presented with a word processor file showing the entire Lambda genome. This enables you to simulate theoretically the same restrictions which you are carrying out practically with The Lambda DNA protocol.

1.	Place the disc in the disc drive.
2.	Open from the disc the file called uncut.doc.
3.	Browse through the Lambda genome.
4.	Estimate the number of bases in the sequence.

(The other file on the disc, master.doc is the entire Lambda DNA sequence and serves as a back-up copy of the genome. It is best not to load this so that there is no danger of altering it.)

The Lambda base sequence may be cut at various points with restriction enzymes. As we have seen each enzyme cuts within a particular base sequence; e.g., EcoRI cuts between the A and the G in the six base sequence GAATTC and nowhere else. This is usually shown as G | AATTC. Other restriction sites are BamHI - G | GATCC and HindIII - A | AGCTT. You can use this word processor file of the Lambda base sequence to find the restriction sites of these three enzymes - and indeed any other enzymes for which you know the 'recognition' sequence. You can then 'cut the genome' and count the number of fragments and the lengths of each of these fragments. These can later be compared with the results of your gel electrophoresis runs from The Lambda DNA protocol.

5.	Place the cursor at the beginning of the base sequence.
6.	Open the EDIT window and select REPLACE.
7.	In answer to the question 'Find what ?' type in gaattc.
8.	Move the cursor to 'Replace with ?' and type in G^pAATTC.
9.	Select REPLACE ALL.


	This cuts the base sequence at all the EcoRI restriction sites and puts a paragraph break (^p) at each point where the sequence has been cut. It also converts the recognition sequence of six bases to upper case letters so that you can see them more easily.

QUESTIONS

a.	How many restriction sites were there ?
b.	How many resulting fragments will there be ?

10.	Select OK and then choose CLOSE.
11.	Highlight the first 'paragraph' of the genome (in reality the first fragment resulting from restriction) by double clicking anywhere in the margin alongside the first few lines.
12.	Open the TOOLS window and select WORDCOUNT. Note the number of characters (that is, the number of bases) in this fragment and enter this number alongside the first fragment below:


	EcoRI - restriction site G \| AATTC

13.	Now select CANCEL.
14.	Move the cursor to the start of the next paragraph, highlight as before, and repeat - noting the number of characters (that is, bases) alongside the next fragment in the bar shown above.
15.	Repeat this process until you reach the end of the genome.


	You should now have the number of fragments you noted in answer to the second question above and shown on the EcoRI bar. You should also have the fragment lengths expressed as the number of bases.

16.	Now arrange the fragments resulting from the restriction by EcoRI in order of size, starting with the largest. Enter these in the table below.
17.	Close the file remembering to answer NO to the question 'Do you want to save changes to 'UNCUT.WORD ?'
18.	Repeat the whole process for BamHI and HindIII, in each case entering the fragment sizes below under the appropriate bars below:


	BamHI - restriction site G \| GATCC


	HindIII - restriction site A \| AGCTT

19.	Close the file remembering to answer NO to the question 'Do you want to save changes to UNCUT.DOC ?'
20.	Now arrange your fragments resulting from the restriction by BamHI and HindIII in order of size, starting with the largest and enter these in the table below alongside the values for EcoRI:

Restriction enzyme	Largest fragment	Smallest fragment	Total number of fragments
EcoRI
BamHI
HindIII

21.	Compare all three sets of fragments in the table with your gels and identify as many of the bands as you can.


	Copyright © National Centre for Biotechnology Education, 2008 \| www.ncbe.reading.ac.uk