diff --git a/src/OpenSSL/SSL.py b/src/OpenSSL/SSL.py
index b4aa78c8..e10c6a62 100644
--- a/src/OpenSSL/SSL.py
+++ b/src/OpenSSL/SSL.py
@@ -835,13 +835,9 @@ def _require_not_used(f: F) -> F:
     @wraps(f)
     def inner(self: Context, *args: Any, **kwargs: Any) -> Any:
         if self._used:
-            warnings.warn(
-                (
-                    "Attempting to mutate a Context after a Connection was "
-                    "created. In the future, this will raise an exception"
-                ),
-                DeprecationWarning,
-                stacklevel=2,
+            raise ValueError(
+                "Context has already been used to create a Connection, it "
+                "cannot be mutated again"
             )
         return f(self, *args, **kwargs)
 
diff --git a/tests/test_ssl.py b/tests/test_ssl.py
index d97cc42b..083e7024 100644
--- a/tests/test_ssl.py
+++ b/tests/test_ssl.py
@@ -1077,6 +1077,7 @@ def test_set_proto_version(self) -> None:
         with pytest.raises(Error, match="unsupported protocol"):
             self._handshake_test(server_context, client_context)
 
+        client_context = Context(TLS_METHOD)
         client_context.set_max_proto_version(0)
         self._handshake_test(server_context, client_context)
 
@@ -1491,8 +1492,6 @@ def test_set_verify_callback_reference(self) -> None:
             load_certificate(FILETYPE_PEM, root_cert_pem)
         )
 
-        clientContext = Context(TLSv1_2_METHOD)
-
         clients = []
 
         for i in range(5):
@@ -1500,13 +1499,16 @@ def test_set_verify_callback_reference(self) -> None:
             def verify_callback(*args: object) -> bool:
                 return True
 
+            # Create a fresh client context for each iteration since contexts
+            # cannot be mutated after use
+            clientContext = Context(TLSv1_2_METHOD)
+            clientContext.set_verify(VERIFY_PEER, verify_callback)
+
             serverSocket, clientSocket = socket_pair()
             client = Connection(clientContext, clientSocket)
 
             clients.append((serverSocket, client))
 
-            clientContext.set_verify(VERIFY_PEER, verify_callback)
-
         gc.collect()
 
         # Make them talk to each other.
@@ -2835,21 +2837,22 @@ def callback(
         del callback
 
         conn = Connection(context, None)
-        context.set_verify(VERIFY_NONE)
 
         collect()
         collect()
         assert tracker()
 
+        # Setting a new callback on the connection should maintain the original
+        # context callback reference
         conn.set_verify(
             VERIFY_PEER, lambda conn, cert, errnum, depth, ok: bool(ok)
         )
         collect()
         collect()
+
+        # The callback should still be referenced - check that it exists
         callback_ref = tracker()
-        if callback_ref is not None:  # pragma: nocover
-            referrers = get_referrers(callback_ref)
-            assert len(referrers) == 1
+        assert callback_ref is not None
 
     def test_get_session_unconnected(self) -> None:
         """
@@ -3708,12 +3711,10 @@ class TestMemoryBIO:
     Tests for `OpenSSL.SSL.Connection` using a memory BIO.
     """
 
-    def _server(self, sock: socket | None) -> Connection:
+    def _create_server_context(self) -> Context:
         """
-        Create a new server-side SSL `Connection` object wrapped around `sock`.
+        Create a configured server context with certificates and options.
         """
-        # Create the server side Connection.  This is mostly setup boilerplate
-        # - use TLSv1, use a particular certificate, etc.
         server_ctx = Context(SSLv23_METHOD)
         server_ctx.set_options(OP_NO_SSLv2 | OP_NO_SSLv3 | OP_SINGLE_DH_USE)
         server_ctx.set_verify(
@@ -3730,6 +3731,23 @@ def _server(self, sock: socket | None) -> Connection:
         )
         server_ctx.check_privatekey()
         server_store.add_cert(load_certificate(FILETYPE_PEM, root_cert_pem))
+        return server_ctx
+
+    def _server(
+        self, sock: socket | None, ctx: Context | None = None
+    ) -> Connection:
+        """
+        Create a new server-side SSL `Connection` object wrapped around `sock`.
+
+        :param sock: The socket to wrap, or None for memory BIO.
+        :param ctx: Optional pre-configured context. If None, creates a
+            default server context.
+        """
+        if ctx is None:
+            server_ctx = self._create_server_context()
+        else:
+            server_ctx = ctx
+
         # Here the Connection is actually created.  If None is passed as the
         # 2nd parameter, it indicates a memory BIO should be created.
         server_conn = Connection(server_ctx, sock)
@@ -3939,12 +3957,16 @@ def _check_client_ca_list(
             that `get_client_ca_list` returns the proper value at
             various times.
         """
-        server = self._server(None)
+        # Create a server context and configure it before creating connections
+        server_ctx = self._create_server_context()
+
+        # Configure the CA list before creating connections
+        expected = func(server_ctx)
+
+        # Now create connections with the configured context
+        server = self._server(None, server_ctx)
         client = self._client(None)
-        assert client.get_client_ca_list() == []
-        assert server.get_client_ca_list() == []
-        ctx = server.get_context()
-        expected = func(ctx)
+
         assert client.get_client_ca_list() == []
         assert server.get_client_ca_list() == expected
         interact_in_memory(client, server)